Integrated system for processing microfluidic samples, and method of using the same

ABSTRACT

This patent application describes an integrated apparatus for processing polynucleotide-containing samples, and for providing a diagnostic result thereon. The apparatus is configured to receive a microfluidic cartridge that contains reagents and a network for processing a sample. Also described are methods of using the apparatus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/719,692, filed May 22, 2015, which is a continuation of U.S.application Ser. No. 11/728,964, filed Mar. 26, 2007 and issued as U.S.Pat. No. 9,040,288 on May 26, 2015, which claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No.60/786,007, filed Mar. 24, 2006, and 60/859,284, filed Nov. 14, 2006.The disclosures of all of the above-referenced prior applications,publications, and patents are considered part of the disclosure of thisapplication, and are incorporated by reference herein in their entirety.

This application is also related to, and incorporates herein byreference the specifications of U.S. Design application Ser. Nos.29/257,028, 29/257,029, and 29/257,030, all of which filed on Mar. 27,2006, and also U.S. patent application Ser. No. 11/580,267, filed Oct.11, 2006, also incorporated herein by reference.

TECHNICAL FIELD

The technology described herein relates to an integrated apparatus forprocessing polynucleotide-containing samples and carrying out diagnostictests on the same. More specifically, the technology relates to anapparatus for obtaining a diagnostic result on a biological sample usinga microfluidic cartridge that receives the sample, in conjunction with abench-top system. Methods of using the technology are also describedherein.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, there are usually several steps in adiagnostic analysis between collecting the sample, and obtaining adiagnostic result, that require different levels of skill by operators,and different levels of complexity of equipment. For example, abiological sample, once extracted from a patient, must be put in a formsuitable for a processing regime that typically involves usingpolymerase chain reaction (PCR) to amplify a nucleotide of interest.Once amplified, the presence of a nucleotide of interest in the sampleneeds to be determined unambiguously. Sample preparation is a processthat is susceptible to automation but is also relatively routinelycarried out in almost any location. By contrast, steps such as PCR andnucleotide detection have customarily only been within the compass ofspecially trained individuals having access to specialist equipment.Second, many diagnostic analyses can only be done with highly specialistequipment that is both expensive and only operable by trainedclinicians. Such equipment is found in only a few locations—often justone in any given urban area. This means that most hospitals are requiredto send out samples to these locations for analysis, thereby incurringshipping costs and transportation delays, and possibly even sample loss,or mix-up. Third, some specialist equipment is typically not available‘on-demand’ but instead runs in batches, thereby delaying the processingtime for many samples because they must wait for a machine to fill upbefore they can be run.

The analysis of a biological sample to accomplish a particular diagnosistypically includes detecting one or more polynucleotides present in thesample. One example of detection is qualitative detection, whichrelates, for example, to the determination of the presence of thepolynucleotide and/or the determination of information related to, forexample, the type, size, presence or absence of mutations, and/or thesequence of the polynucleotide. Another example of detection isquantitative detection, which relates, for example, to the determinationof the amount of polynucleotide present. Detection may thereforegenerally include both qualitative and quantitative aspects. Detectingpolynucleotides qualitatively often involves establishing the presenceof extremely small quantities in a sample. In order to improvesensitivity, therefore, the amount of polynucleotide in question isoften amplified. For example, some detection methods includepolynucleotide amplification by polymerase chain reaction (PCR) or arelated amplification technique. Such techniques use a cocktail ofingredients, including one or more of an enzyme, a probe, and a labelingagent. Therefore, detection of polynucleotides can require use of avariety of different reagents, many of which require sensitive handlingto maintain their integrity, both during use, and over time.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible, and, desirably, to facilitate accomplishing as many aspossible with a single machine that can be made available, on demand, tomany users. There is therefore need for a method and apparatus ofcarrying out steps of sample preparation, PCR, and detection onbiological samples in such a way that as few separate steps as possibleare carried out.

The discussion of the background to the technology herein is included toexplain the context of the technology. This is not to be taken as anadmission that any of the material referred to was published, known, orpart of the common general knowledge as at the priority date of any ofthe claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

An apparatus, comprising: a receiving bay configured to receive aninsertable microfluidic cartridge; at least one heat source thermallycoupled to the cartridge and configured to apply heat to one or moreselected regions of the cartridge at one or more selected times, inorder to: create a micro-droplet of a polynucleotide-containingbiological sample held on the cartridge; cause the micro-droplet to movebetween one or more positions on the microfluidic cartridge; lyse cells,where present in the biological sample, thereby releasingpolynucleotides from the cells; prepare one or more of thepolynucleotides for amplification; and amplify one or more of thepolynucleotides; a detector configured to detect presence of the one ormore amplified polynucleotides; and a processor coupled to the detectorand the at least one heat source, wherein the processor is configured tocontrol applying heat to the one or more selected regions of themicrofluidic cartridge at one or more selected times.

The system herein further comprises an integrated system, comprising anapparatus and a complementary cartridge, wherein together the apparatusand cartridge process a sample that has been injected into thecartridge, and provide a diagnostic result on the sample.

The receiving bay of the apparatus can be configured to selectivelyreceive the microfluidic cartridge, as further described herein andexemplified by the accompanying drawings. For example, the receiving bayand the microfluidic cartridge can be complementary in shape so that themicrofluidic cartridge can be selectively received in, e.g., a singleorientation. The microfluidic cartridge can have a registration memberthat fits into a complementary feature of the receiving bay. Byselectively receiving the cartridge, the receiving bay can help a userto place the cartridge so that the apparatus can properly operate on thecartridge. The receiving bay can also be configured so that variouscomponents of the apparatus that can operate on the microfluidiccartridge (heat pumps, peltier coolers, heat-removing electronicelements, detectors, force members, and the like) can be positioned toproperly operate on the microfluidic cartridge. For example, a contactheat source can be situated in the receiving bay such that it can bethermally coupled to one or more distinct locations of a microfluidiccartridge that can be selectively received in the receiving bay.

The heat pump can be, for example, a heat source such as a resistor, areversible heat pump such as a liquid-filled heat transfer circuit or athermoelectric element, a radiative heat source such as a xenon lamp,and the like. The heat pump may be used not only to provide heat to themicrofluidic elements but also to remove heat from microfluidic elementssuch as to reduce activity of certain reagents, freeze liquid in amicrochannel to change its phase from liquid to solid, reduce thepressure of an air chamber to create a partial vacuum, etc.)

In various embodiments of the apparatus: the apparatus can furtherinclude a registration member that is complementary to the microfluidiccartridge, whereby the receiving bay receives the microfluidic cartridgein a single orientation; the apparatus can further include a sensorcoupled to a processor, the sensor configured to sense whether themicrofluidic cartridge can be selectively received.

The processor can be programmable to operate the detector to detect apolynucleotide or a probe thereof in a microfluidic cartridge located inthe receiving bay.

The detector can be, for example, an optical detector. For example, thedetector can include a light source that emits light in an absorptionband of a fluorescent dye and a light detector that detects light in anemission band of the fluorescent dye, wherein the fluorescent dyecorresponds to a fluorescent polynucleotide probe or a fragment thereof.For example, the optical detector can include a bandpass-filtered diodethat selectively emits light in the absorption band of the fluorescentdye and a bandpass filtered photodiode that selectively detects light inthe emission band of the fluorescent dye; or for example, the opticaldetector can be configured to independently detect a plurality offluorescent dyes having different fluorescent emission spectra, whereineach fluorescent dye corresponds to a fluorescent polynucleotide probeor a fragment thereof; or for example, the optical detector can beconfigured to independently detect a plurality of fluorescent dyes at aplurality of different locations in the cartridge, wherein eachfluorescent dye corresponds to a fluorescent polynucleotide probe or afragment thereof.

The processor can be, for example, programmable to operate the at leastone heat pump.

In various embodiments, the at least one heat pump can be a contact heatsource selected from a resistive heater, a radiator, a fluidic heatexchanger and a Peltier device. The contact heat source can beconfigured at the receiving bay to be thermally coupled to a distinctlocation in a microfluidic cartridge received in the receiving bay,whereby the distinct location can be selectively heated. At least oneadditional contact heat source can be included, wherein the contact heatsources can be each configured at the receiving bay to be independentlythermally coupled to a different distinct location in a microfluidiccartridge received in the receiving bay, whereby the distinct locationscan be independently heated. The contact heat source can be configuredto be in direct physical contact with a distinct location of amicrofluidic cartridge received in the receiving bay. In variousembodiments, each contact source heater can be configured to heat adistinct location having an average diameter in 2 dimensions from about1 millimeter (mm) to about 15 mm (typically about 1 mm to about 10 mm),or a distinct location having a surface area of between about 1 mm²about 225 mm² (typically between about 1 mm² and about 100 mm², or insome embodiments between about 5 mm² and about 50 mm²).

In various embodiments, the apparatus can include a compliant layer atthe contact heat source, configured to thermally couple the contact heatsource with at least a portion of a microfluidic cartridge received inthe receiving bay. The compliant layer can have a thickness of betweenabout 0.05 and about 2 millimeters, and a Shore hardness of betweenabout 25 and about 100.

In various embodiments, at least one heat pump can be a radiative heatsource configured to direct heat to a distinct location of amicrofluidic cartridge received in the receiving bay.

In various embodiments, the one or more force members configured toapply force to at least a portion of a microfluidic cartridge receivedin the receiving bay.

In various embodiments, the one or more force members can be configuredto apply force to thermally couple the at least one heat pump to atleast a portion of the microfluidic cartridge. The one or more forcemembers can be configured to operate a mechanical member at themicrofluidic cartridge, the mechanical member selected from the groupconsisting of a pierceable reservoir, a valve or a pump.

In various embodiments, the one or more force members can be configuredto apply force to a plurality of locations in the microfluidiccartridge. The force applied by the one or more force members can resultin an average pressure at an interface between a portion of thereceiving bay and a portion of the microfluidic cartridge of betweenabout 5 kilopascals and about 50 kilopascals, for example, the averagepressure can be at least about 14 kilopascals. At least one force membercan be manually operated. At least one force member can be mechanicallycoupled to a lid at the receiving bay, whereby operation of the lidoperates the force member.

In various embodiments, the apparatus can further include a lid at thereceiving bay, the lid being operable to at least partially excludeambient light from the receiving bay. The lid can be, for example, asliding lid. The lid can include the optical detector. A major face ofthe lid at the optical detector or at the receiving bay can vary fromplanarity by less than about 100 micrometers, for example, less thanabout 25 micrometers. The lid can be configured to be removable from theapparatus. The lid can include a latching member.

In various embodiments, the apparatus can further include at least oneinput device coupled to the processor.

In various embodiments, the apparatus can further include a heatingstage configured to be removable from the apparatus wherein at least oneheat pump can be located in the heating stage.

In various embodiments, the cartridge can further include an analysisport. The analysis port can be configured to allow an external samplesystem to analyze a sample in the microfluidic cartridge; for example,the analysis port can be a hole or window in the apparatus which canaccept an optical detection probe that can analyze a sample in situ inthe microfluidic cartridge.

In some embodiments, the analysis port can be configured to direct asample from the microfluidic cartridge to an external sample system; forexample, the analysis port can include a conduit in fluid communicationwith the microfluidic cartridge that directs a liquid sample to achromatography apparatus, an optical spectrometer, a mass spectrometer,or the like.

In some embodiments, the apparatus can include a receiving bayconfigured to receive a microfluidic cartridge in a single orientation;at least one radiative heat source thermally coupled to the receivingbay; at least two contact heat sources configured in the receiving bayto be thermally coupled to distinct locations, whereby the distinctlocations can be selectively heated; one or more force membersconfigured to apply force to at least a portion of the microfluidiccartridge received in the receiving bay, wherein at least one of the oneor more force members can be configured to apply force to thermallycouple the contact heat sources to the distinct locations, and at leastone of the one or more force members can be configured to operate amechanical member at the microfluidic cartridge, the mechanical memberselected from the group consisting of a pierceable reservoir; a lid atthe receiving bay, the lid being operable to at least partially excludeambient light from the receiving bay, the lid comprising an opticaldetector configured to independently detect one or more fluorescentdyes, optionally having different fluorescent emission spectra, whereineach fluorescent dye corresponds to a fluorescent polynucleotide probeor a fragment thereof; at least one input device selected from the groupconsisting of a keyboard, a touch-sensitive surface, a microphone, and amouse, at least one data storage medium selected from the groupconsisting of a hard disk drive, an optical disk drive, a communicationinterface selected from the group consisting of: a serial connection, aparallel connection, a wireless network connection, and a wired networkconnection, a sample identifier selected from an optical characterreader, a bar code reader, and a radio frequency tag reader; at leastone output selected from a display, a printer, a speaker; and aprocessor coupled to the detector, the sensor, the heat sources, theinput, and the output.

A microfluidic cartridge can include a microfluidic network and aretention member in fluid communication with the microfluidic network,the retention member being selective for at least one polynucleotideover at least one polymerase chain reaction inhibitor. In someembodiment, the microfluidic cartridge also includes a registrationmember.

In various embodiments of the microfluidic cartridge, the microfluidiccartridge can further include a sample inlet valve in fluidcommunication with the microfluidic network. The sample inlet valve canbe configured to accept a sample at a pressure differential compared toambient pressure of between about 20 kilopascals and 200 kilopascals,for example between about 70 kilopascals and 110 kilopascals.

In various embodiments, the microfluidic network can include a filter influid communication with the sample inlet valve, the filter beingconfigured to separate at least one component from a sample mixtureintroduced at the sample inlet.

In various embodiments, the microfluidic network can include at leastone thermally actuated pump in fluid communication with the microfluidicnetwork. The thermally actuated pump can include a thermoexpansivematerial selected from a gas, a liquid vaporizable at a temperaturebetween 25° C. and 100° C. at 1 atmosphere, and an expancel polymer.

In various embodiments, the microfluidic network can include at leastone thermally actuated valve in fluid communication with themicrofluidic network. The thermally actuated valve can include amaterial having a solid to liquid phase transition at a temperaturebetween 25° C. and 100° C. at 1 atmosphere.

In various embodiments, the microfluidic network can include at leastone sealed reservoir containing a reagent, a buffer or a solvent. Thesealed reservoir can be, for example, a self-piercing blister packconfigured to bring the reagent, the buffer or the solvent into fluidcommunication with the microfluidic network.

In various embodiments, the microfluidic network can include at least atleast one hydrophobic vent.

In various embodiments, the microfluidic network can include at leastone reservoir configured to receive and to contain waste such as fluidsand/or particulate matter such as cellular debris.

In various embodiments, the retention member can include a polyalkyleneimine or a polycationic polyamide, for example, polyethylene imine,poly-L-lysine or poly-D-lysine. The retention member can be in the formof one or more particles. The retention member can be removable from themicrofluidic cartridge.

In various embodiments, the microfluidic network can include a lysisreagent. The lysis reagent can include one or more lyophilized pelletsof surfactant, wherein the microfluidic network can be configured tocontact the lyophilized pellet of surfactant with a liquid to create alysis reagent solution. The microfluidic network can be configured tocontact a sample with the lysis reagent to produce a lysed sample.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source to the sample to produce thelysed sample. For example, the microfluidic network can be configured tocontact the retention member and the lysed sample to create apolynucleotide-loaded retention member.

In various embodiments, the microfluidic cartridge can further include afilter configured to separate the polynucleotide-loaded retention memberfrom liquid.

In various embodiments, the microfluidic cartridge can further include areservoir containing a wash buffer, wherein the microfluidic network canbe configured to contact the polynucleotide-loaded retention member withthe wash buffer, for example, the wash buffer can have a pH of at leastabout 10.

In various embodiments, the microfluidic cartridge can include areservoir containing a release buffer, wherein the microfluidiccartridge can be configured to contact the polynucleotide-loadedretention member with the release buffer to create a releasedpolynucleotide sample.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source to the polynucleotide-loadedretention member to create the released polynucleotide sample.

In various embodiments, the microfluidic cartridge can include areservoir containing a neutralization buffer, wherein the microfluidicnetwork can be configured to contact the released polynucleotide samplewith the neutralization buffer to create a neutralized polynucleotidesample.

In various embodiments, the microfluidic cartridge can include a PCRreagent mixture comprising a polymerase enzyme and a plurality ofnucleotides. The PCR reagent mixture can be in the form of one or morelyophilized pellets, and the microfluidic network can be configured tocontact the PCR pellet with liquid to create a PCR reagent mixturesolution.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source with the PCR reagent mixtureand the neutralized polynucleotide sample under thermal cyclingconditions suitable for creating PCR amplicons from the neutralizedpolynucleotide sample.

In various embodiments, the PCR reagent mixture can further include apositive control plasmid and a fluorogenic hybridization probe selectivefor at least a portion of the plasmid.

In various embodiments, the microfluidic cartridge can include anegative control polynucleotide, wherein the microfluidic network can beconfigured to independently contact each of the neutralizedpolynucleotide sample and the negative control polynucleotide with thePCR reagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide.

In various embodiments, the microfluidic cartridge can include at leastone probe that can be selective for a polynucleotide sequence, whereinthe microfluidic cartridge can be configured to contact the neutralizedpolynucleotide sample or a PCR amplicon thereof with the probe. Theprobe can be a fluorogenic hybridization probe. The fluorogenichybridization probe can include a polynucleotide sequence coupled to afluorescent reporter dye and a fluorescence quencher dye. The PCRreagent mixture can further include a positive control plasmid and aplasmid fluorogenic hybridization probe selective for at least a portionof the plasmid and the microfluidic cartridge can be configured to allowindependent optical detection of the fluorogenic hybridization probe andthe plasmid fluorogenic hybridization probe.

In various embodiments, the probe can be selective for a polynucleotidesequence that can be characteristic of an organism, for example anyorganism that employs deoxyribonucleic acid or ribonucleic acidpolynucleotides. Thus, the probe can be selective for any organism.Suitable organisms include mammals (including humans), birds, reptiles,amphibians, fish, domesticated animals, farmed animals, wild animals,extinct organisms, bacteria, fungi, viruses, plants, and the like. Theprobe can also be selective for components of organisms that employtheir own polynucleotides, for example mitochondria. In someembodiments, the probe can be selective for microorganisms, for example,organisms used in food production (for example, yeasts employed infermented products, molds or bacteria employed in cheeses, and the like)or pathogens (e.g., of humans, domesticated or wild mammals,domesticated or wild birds, and the like). In some embodiments, theprobe can be selective for organisms selected from the group consistingof gram positive bacteria, gram negative bacteria, yeast, fungi,protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of Group B Streptococcus.

In various embodiments, the microfluidic cartridge can be configured toallow optical detection of the fluorogenic hybridization probe.

In various embodiments, the microfluidic cartridge can further include acomputer-readable label. For example, the label can include a bar code,a radio frequency tag or one or more computer-readable characters. Thelabel can be formed of a mechanically compliant material. For example,the mechanically compliant material of the label can have a thickness ofbetween about 0.05 and about 2 millimeters and a Shore hardness ofbetween about 25 and about 100.

In various embodiments, the microfluidic cartridge can be furthersurrounded by a sealed pouch, during handling and storage, and prior tobeing inserted into the chamber. The microfluidic cartridge can besealed in the pouch with an inert gas. The sealed pouch may also containa packet of dessicant. The microfluidic cartridge can be disposable.

In various embodiments, the microfluidic cartridge can contain one ormore sample lanes. For example, a sample lane can include a thermallyactuated pump, a thermally actuated valve, a sample inlet valve, afilter, and at least one reservoir. The lanes can be independent of eachother, or can be partially dependent, for example, the lanes can shareone or more reagents such as the lysis reagent.

In some embodiments, the microfluidic cartridge can include aregistration member; and a microfluidic network. The microfluidicnetwork includes, in fluidic communication: at least one thermallyactuated pump; at least one thermally actuated valve; a sample inletvalve configured to accept a sample at a pressure differential comparedto ambient pressure of between about 70 kilopascals and 110 kilopascals;a retention member selective for at least one polynucleotide over atleast one polymerase chain reaction inhibitor, the retention memberbeing in the form of a plurality of particles formed of a polyalkyleneimine or a polycationic polyamide; a filter configured to separate thepolynucleotide-loaded retention member from liquid; a plurality ofreservoirs, at least said one said reservoir being a sealed,self-piercing blister pack reservoir. The plurality of reservoirs cancontain among them: a lysis reagent, the microfluidic network beingconfigured to contact a sample introduced at the sample inlet with thelysis reagent and the retention member to create a polynucleotide-loadedretention member; a reservoir containing a wash buffer, the microfluidicnetwork being configured to contact the polynucleotide-loaded retentionmember with the wash buffer; a reservoir containing a release buffer,the microfluidic network being configured to contact thepolynucleotide-loaded retention member with the release buffer to createa released polynucleotide sample; a neutralization buffer, themicrofluidic network being configured to contact the releasedpolynucleotide sample with the neutralization buffer to create aneutralized polynucleotide sample; a PCR reagent mixture comprising apolymerase enzyme, a positive control plasmid, a fluorogenichybridization probe selective for at least a portion of the plasmid anda plurality of nucleotides; and at least one probe that can be selectivefor a polynucleotide sequence, wherein the microfluidic network can beconfigured to contact the neutralized polynucleotide sample or a PCRamplicon thereof with the probe. Further, the microfluidic network canbe configured to couple heat from an external heat source with the PCRreagent mixture and the neutralized polynucleotide sample under thermalcycling conditions suitable for creating PCR amplicons from theneutralized polynucleotide sample.

In various embodiments, a polynucleotide analysis system can includeboth the the microfluidic cartridge and the apparatus, as furtherdescribed herein.

In various embodiments, a polynucleotide sample kit can include amicrofluidic cartridge comprising a microfluidic network and a retentionmember in fluid communication with the microfluidic network, theretention member being selective for at least one polynucleotide over atleast one polymerase chain reaction inhibitor a sample container; and aliquid transfer member such as a syringe.

In various embodiments, the polynucleotide sample kit can furtherinclude instructions to employ the liquid transfer member to transfer asample from the sample container to the microfluidic network.

In various embodiments, the polynucleotide sample kit can furtherinclude instructions to employ the liquid transfer member to direct asample from the sample container, and a volume of air into themicrofluidic network, the volume of air being between about 0.5 mL andabout 5 mL.

In various embodiments, the polynucleotide sample kit can furtherinclude a filter, and, for example, instructions to employ the liquidtransfer member to direct a sample from the sample container through thefilter into the microfluidic network.

In various embodiments, the polynucleotide sample kit can furtherinclude at least one computer-readable label on the sample container.The label can include, for example, a bar code, a radio frequency tag orone or more computer-readable characters. The microfluidic cartridge canbe sealed in a pouch with an inert gas.

In various embodiments, the polynucleotide sample kit can furtherinclude a sampling member; a transfer container; and instructions tocontact the sampling member to a biological sample and to place thesampling member in the transfer container.

In various embodiments, the polynucleotide sample kit can furtherinclude a sample buffer, and, for example, instructions to contact thesampling member and the sample buffer.

In various embodiments, the polynucleotide sample kit can furtherinclude at least one probe that can be selective for a polynucleotidesequence, e.g., the polynucleotide sequence that is characteristic of apathogen selected from the group consisting of gram positive bacteria,gram negative bacteria, yeast, fungi, protozoa, and viruses.

In some embodiments, the polynucleotide sample kit can include amicrofluidic cartridge comprising a microfluidic network, a retentionmember in fluid communication with the microfluidic network, and afluorogenic probe, the retention member being selective for at least onepolynucleotide over at least one polymerase chain reaction inhibitor,the fluorogenic probe being selective for a polynucleotide sequence thatcan be characteristic of a pathogen selected from the group consistingof gram positive bacteria, gram negative bacteria, yeast, fungi,protozoa, and viruses; a sample container; a liquid transfer member; asampling member; a transfer container; a sample buffer; andinstructions. The instructions can include instructions to: employ theliquid transfer member to transfer a sample from the sample container tothe microfluidic network; employ the liquid transfer member to direct asample from the sample container and a volume of air into themicrofluidic network, the volume of air being between about 0.5 mL andabout 5 mL; employ the liquid transfer member to direct a sample fromthe sample container through a filter into the microfluidic network; andcontact the sampling member to a biological sample and to place thesampling member in the transfer container.

A method for sampling a polynucleotide can include the steps ofcontacting the retention member at the microfluidic cartridge with abiological sample, the biological sample comprising at least onepolynucleotide, thereby producing a polynucleotide-loaded retentionmember in the microfluidic cartridge; separating at least a portion ofthe biological sample from the polynucleotide-loaded retention member;and releasing at least a portion of a polynucleotide from thepolynucleotide-loaded retention member, thereby creating a releasedpolynucleotide sample.

In various embodiments, the method can further include one or more ofthe following steps: placing the microfluidic cartridge in the receivingbay of the apparatus; operating the force member in the apparatus toapply pressure at an interface between a portion of the receiving bayand a portion of the microfluidic cartridge (e.g., creating a pressurebetween about 5 kilopascals and about 50 kilopascals, or in someembodiments, at least about 14 kilopascals); employing the force memberto apply force to a mechanical member in the microfluidic cartridge, themechanical member selected from the group consisting of a pierceablereservoir, a valve or a pump, to release at least one reagent, buffer,or solvent from a reservoir in the microfluidic chip; and/or closing thelid to operate the force member, wherein the force member can bemechanically coupled to a lid at the receiving bay.

In some embodiments, the method can further include employing a sampleidentifier to read a label on the microfluidic cartridge or a label onthe biological sample.

In some embodiments, the method can further include introducing a crudebiological sample into the microfluidic cartridge and separating thebiological sample from the crude biological sample in the microfluidiccartridge, e.g., using a filter in the cartridge, or the biologicalsample can be separated from a crude biological sample prior tointroducing the biological sample into the microfluidic cartridge.

In some embodiments, the method can further include lysing thebiological sample, for example, using heat, a lysis reagent, and thelike. In some embodiments, wherein the microfluidic cartridge comprisesone or more lyophilized pellets of lysis reagent, the method can furtherinclude reconstituting the lyophilized pellet of surfactant with liquidto create a lysis reagent solution.

In various embodiments, the method can further include one or more ofthe following: heating the biological sample in the microfluidiccartridge; pressurizing the biological sample in the microfluidiccartridge at a pressure differential compared to ambient pressure ofbetween about 20 kilopascals and 200 kilopascals, or in some embodimentsbetween about 70 kilopascals and 110 kilopascals.

In some embodiments, the portion of the biological sample separated fromthe polynucleotide-loaded retention member can include at least onepolymerase chain reaction inhibitor selected from the group consistingof hemoglobin, peptides, faecal compounds, humic acids, mucousolcompounds, DNA binding proteins, or a saccharide. In some embodiments,the method can further include separating the polynucleotide-loadedretention member from substantially all of the polymerase chain reactioninhibitors in the biological sample.

In various embodiments, the method can further include one or more ofthe following: directing a fluid in the microfluidic cartridge byoperating a thermally actuated pump or a thermally actuated valve;contacting the polynucleotide-loaded retention member with a washbuffer; heating the polynucleotide-loaded retention member to atemperature of at least about 50° C. (in some embodiments, thetemperature can be 100° C. or less); heating the polynucleotide-loadedretention member for less than about 10 minutes; contacting thepolynucleotide-loaded retention member with a release buffer to create areleased polynucleotide sample (for example, in some embodiments, thethe release buffer can have a volume of less than about 50 microliters,the release buffer can include a detergent, and/or the release buffercan have a pH of at least about 10); and/or contacting the releasedpolynucleotide sample with a neutralization buffer to create aneutralized polynucleotide sample.

In various embodiments, the method can further include one or more ofthe following: contacting the neutralized polynucleotide sample with aPCR reagent mixture comprising a polymerase enzyme and a plurality ofnucleotides (in some embodiments, the PCR reagent mixture can furtherinclude a positive control plasmid and a fluorogenic hybridization probeselective for at least a portion of the plasmid); in some embodiments,the PCR reagent mixture can be in the form of one or more lyophilizedpellets, and the method can further include reconstituting the PCRpellet with liquid to create a PCR reagent mixture solution; heating thePCR reagent mixture and the neutralized polynucleotide sample underthermal cycling conditions suitable for creating PCR amplicons from theneutralized polynucleotide sample; contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof with at least one probethat can be selective for a polynucleotide sequence; independentlycontacting each of the neutralized polynucleotide sample and a negativecontrol polynucleotide with the PCR reagent mixture under thermalcycling conditions suitable for independently creating PCR amplicons ofthe neutralized polynucleotide sample and PCR amplicons of the negativecontrol polynucleotide; and/or contacting the neutralized polynucleotidesample or a PCR amplicon thereof and the negative control polynucleotideor a PCR amplicon thereof with at least one probe that is selective fora polynucleotide sequence.

In various embodiments, the method can further include one or more ofthe following: determining the presence of a polynucleotide sequence inthe biological sample, the polynucleotide sequence corresponding to theprobe, if the probe is detected in the neutralized polynucleotide sampleor a PCR amplicon thereof; determining a contaminated result if theprobe is detected in the negative control polynucleotide or a PCRamplicon thereof; and/or in some embodiments, wherein wherein the PCRreagent mixture further comprises a positive control plasmid and aplasmid probe selective for at least a portion of the plasmid, themethod further including determining a PCR reaction has occurred if theplasmid probe is detected.

In various embodiments, the method does not comprise centrifugation ofthe polynucleotide-loaded retention member.

In some embodiments, the method for sampling a polynucleotide caninclude: placing a microfluidic cartridge in the receiving bay of anapparatus; operating a force member in the apparatus to apply pressureat an interface between a portion of the receiving bay and a portion ofthe microfluidic cartridge, the force operating to release at least onereagent, buffer, or solvent from a reservoir in the microfluidiccartridge; lysing a biological sample in the microfluidic cartridge tocreate a lysed biological sample; contacting a retention member at amicrofluidic cartridge with the lysed biological sample, the biologicalsample comprising at least one polynucleotide, thereby producing apolynucleotide-loaded retention member in the microfluidic cartridge,wherein the retention member is in the form of a plurality of particlesof a polyalkylene imine or a polycationic polyamide; contacting thepolynucleotide-loaded retention member with a wash buffer; heating thepolynucleotide-loaded retention member to a temperature of at leastabout 50° C. for less than about 10 minutes; contacting thepolynucleotide-loaded retention member with a release buffer to create areleased polynucleotide sample. contacting the released polynucleotidesample with a neutralization buffer to create a neutralizedpolynucleotide sample; contacting the neutralized polynucleotide samplewith a PCR reagent mixture under thermal cycling conditions suitable forcreating PCR amplicons from the neutralized polynucleotide sample, thePCR reagent mixture comprising a polymerase enzyme, a positive controlplasmid a fluorogenic hybridization probe selective for at least aportion of the plasmid, and a plurality of nucleotides. contacting theneutralized polynucleotide sample or a PCR amplicon thereof with atleast one fluorogenic probe that can be selective for a polynucleotidesequence, wherein the probe can be selective for a polynucleotidesequence that can be characteristic of an organism selected from thegroup consisting of gram positive bacteria, gram negative bacteria,yeast, fungi, protozoa, and viruses; and detecting the fluorogenic probeand determining the presence of the organism for which the onefluorogenic probe can be selective.

In various embodiments, a computer program product includes computerreadable instructions thereon for operating the apparatus.

In some embodiments, a computer program product includes computerreadable instructions thereon for causing the system to create areleased polynucleotide sample from a biological sample. The computerreadable instructions can include instructions for contacting theretention member with the biological sample under conditions suitablefor producing a polynucleotide-loaded retention member; separating atleast a portion of the biological sample from the polynucleotide-loadedretention member; and releasing at least a portion of a polynucleotidefrom the polynucleotide-loaded retention member, thereby creating areleased polynucleotide sample.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: output an indicator of theplacement of the microfluidic cartridge in the receiving bay; read asample label or a microfluidic cartridge label; output directions for auser to input a sample identifier; output directions for a user to loada sample transfer member with the biological sample; output directionsfor a user to apply a filter to the sample transfer member; outputdirections for a user to introduce the biological sample into themicrofluidic cartridge; output directions for a user to cause thebiological sample to contact a lysis reagent in the microfluidiccartridge; output directions for a user to place the microfluidiccartridge in the receiving bay; output directions for a user to operatea force member in the apparatus to apply pressure at an interfacebetween a portion of the receiving bay and a portion of the microfluidiccartridge; output directions for a user to close the lid to operate theforce member; and/or output directions for a user to pressurize thebiological sample in the microfluidic cartridge by injecting thebiological sample with a volume of air between about 0.5 mL and about 5mL.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: lyse the biological sample;lyse the biological sample with a lysis reagent; reconstitute alyophilized pellet of surfactant with liquid to create a lysis reagentsolution; heat the biological sample; separate the polynucleotide-loadedretention member from at least a portion of the biological sample;separate the polynucleotide-loaded retention member from substantiallyall of the polymerase chain reaction inhibitors in the biologicalsample; direct a fluid in the microfluidic cartridge by operating athermally actuated pump or a thermally actuated valve; contact thepolynucleotide-loaded retention member with a wash buffer; heat thepolynucleotide-loaded retention member to a temperature of at leastabout 50° C. (in some embodiments, the temperature can be about 100° C.or less); heat the polynucleotide-loaded retention member for less thanabout 10 minutes; contact the polynucleotide-loaded retention memberwith a release buffer to create a released polynucleotide sample; and/orcontact the released polynucleotide sample with a neutralization bufferto create a neutralized polynucleotide sample.

In various embodiments, the computer program product can include one ormore instructions to cause the system to: contact the neutralizedpolynucleotide sample with a PCR reagent mixture comprising a polymeraseenzyme and a plurality of nucleotides; heat the PCR reagent mixture andthe neutralized polynucleotide sample under thermal cycling conditionssuitable for creating PCR amplicons from the neutralized polynucleotidesample; contact the neutralized polynucleotide sample or a PCR ampliconthereof with at least one probe that can be selective for apolynucleotide sequence; independently contact each of the neutralizedpolynucleotide sample and a negative control polynucleotide with the PCRreagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide; contactthe neutralized polynucleotide sample or a PCR amplicon thereof and thenegative control polynucleotide or a PCR amplicon thereof with at leastone probe that can be selective for a polynucleotide sequence; output adetermination of the presence of a polynucleotide sequence in thebiological sample, the polynucleotide sequence corresponding to theprobe, if the probe is detected in the neutralized polynucleotide sampleor a PCR amplicon thereof; and/or output a determination of acontaminated result if the probe is detected in the negative controlpolynucleotide or a PCR amplicon thereof.

In various embodiments, the computer program product can include one ormore instructions to cause the system to automatically conduct one ormore of the steps of the method.

In various embodiments, wherein the microfluidic network comprises twoor more sample lanes each including a thermally actuated pump, athermally actuated valve, a sample inlet valve, a filter, and at leastone reservoir, wherein the computer readable instructions can beconfigured to independently operate each said lane in the system.

In some embodiments, the computer program product includes computerreadable instructions thereon for causing a system to create a releasedpolynucleotide sample from a biological sample. The system can include amicrofluidic cartridge comprising a microfluidic network and a retentionmember in fluid communication with the microfluidic network, theretention member being selective for at least one polynucleotide over atleast one polymerase chain reaction inhibitor; and an apparatuscomprising a receiving bay configured to selectively receive themicrofluidic cartridge; at least one heat pump configured to bethermally coupled to the microfluidic cartridge in the receiving bay; adetector; and a programmable processor coupled to the detector and theheat pump. The computer readable instructions can include instructionsfor: lysing a biological sample by contacting the biological sample witha lysis reagent and heating to produce a lysed sample; contacting theretention member with the biological sample and/or the lysed sample toproduce a polynucleotide-loaded retention member; separating at least aportion of the biological sample from the polynucleotide-loadedretention member; contacting the polynucleotide-loaded retention memberwith a wash buffer; contacting the polynucleotide-loaded retentionmember with a release buffer and or heat to release at least a portionof a polynucleotide from the polynucleotide-loaded retention member,thereby creating a released polynucleotide sample; contacting thereleased polynucleotide sample with a neutralization buffer to create aneutralized polynucleotide sample; independently contacting each of theneutralized polynucleotide sample and a negative control polynucleotidewith a PCR reagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons, the PCR reagent mixture comprisinga polymerase enzyme, a plurality of nucleotides, a positive controlplasmid and a plasmid probe selective for at least a portion of theplasmid; determining a PCR reaction has occurred if the plasmid probe isdetected; contacting the neutralized polynucleotide sample or a PCRamplicon thereof and the negative control polynucleotide or a PCRamplicon thereof with at least one probe that is selective for apolynucleotide sequence; determining the presence of a polynucleotidesequence in the biological sample, the polynucleotide sequencecorresponding to the probe, if the probe is detected in the neutralizedpolynucleotide sample or a PCR amplicon thereof; and determining acontaminated result if the probe is detected in the negative controlpolynucleotide or a PCR amplicon thereof.

The details of one or more embodiments of the technology are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the technology will be apparent from thedescription and drawings, and from the claims. Like reference symbols inthe various drawings indicate like elements.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic overview of an apparatus described herein.

FIGS. 2A-2E show perspective views of an exemplary apparatus, in variousconfigurations, as further described herein.

FIG. 3 is an exploded view of typical components of an apparatus.

FIG. 4 is a block diagram of an apparatus.

FIG. 5 is a diagram of a fluorescent detection module.

FIGS. 6A and 6B depict the location, in various embodiments, of amicrofluidic cartridge after installation into an apparatus.

FIG. 7 shows a perspective view of a removable heater module, as furtherdescribed herein.

FIGS. 8A-8C show a plan view of heater circuitry adjacent to a PCRreaction zone; and thermal images of heater circuitry in operation.

FIG. 9 shows a perspective view of a microfluidic cartridge, as furtherdescribed herein.

FIG. 10 is a perspective view of a microfluidic device.

FIG. 11 is a cross-sectional view of a processing region for retainingpolynucleotides and/or separating polynucleotides from inhibitors.

FIG. 12A is a perspective view of a gate.

FIG. 12B is a perspective view of a bent gate.

FIG. 13 is a cross-sectional view of an actuator.

FIG. 14A is a perspective view of a microfluidic cartridge.

FIG. 14B is a side cross-sectional view of the microfluidic cartridge ofFIGS. 14A and 14B.

FIGS. 15A and 15B, taken together, illustrate a perspective view of amicrofluidic network of the microfluidic cartridge of FIGS. 14A and 14B.

FIG. 16 illustrates an array of heat sources for operating components ofthe microfluidic cartridge of FIGS. 14A and 14B.

FIGS. 17 and 18 illustrate a valve in the open and closed statesrespectively.

FIG. 19A-19D illustrate a mixing gate of the microfluidic network ofFIGS. 6A and 6B and adjacent regions of the network.

FIGS. 20A-20C illustrate a reservoir with actuation mechanism.

FIGS. 21A-21C illustrate a reservoir with actuation mechanism.

FIG. 22 illustrates a reservoir with actuation mechanism.

FIGS. 23A-23B illustrate a reservoir with actuation mechanism.

FIGS. 24A-24B illustrate a reservoir with actuation mechanism.

FIG. 25 illustrates a reservoir with actuation mechanism.

FIG. 26 illustrates a reservoir with actuation mechanism.

FIGS. 27A and 27B illustrate embodiments of a reagent pack with apiercing member.

FIG. 28 depicts an apparatus that can operate as a small bench-top, realtime polynucleotide analysis system.

FIG. 29 depicts a sample kit that can be used with an apparatus.

FIG. 30 depicts a pouch for components of a sample kit.

FIG. 31 depicts an exemplary microfluidic cartridge.

FIG. 32 depicts the use of a bar code reader to a read bar code on amicrofluidic cartridge.

FIG. 33 depicts the use of a bar code reader a to read a bar code on asample container.

FIG. 34 depicts attachment of a filter to a lysis reservoir of amicrofluidic cartridge via a sample inlet, whereby the contents ofsyringe may be injected into microfluidic cartridge through a filter.

FIG. 35 depicts the addition of air to syringe in order to pressurizemicrofluidic cartridge.

FIG. 36 depicts the placement of a microfluidic cartridge in a receivingbay of an apparatus.

FIGS. 37 and 38 depict the closure of a lid of an apparatus.

FIGS. 39 & 40 depict the removal of a heating/sensor module from anapparatus.

FIG. 41A is a graph of real time heat sensor data from an apparatus.

FIG. 41B is a graph of real time optical detector data from anapparatus.

FIG. 42 is a schematic representation of various exemplary chambersand/or subunits in an exemplary microfluidic cartridge.

FIG. 43 is a schematic representation of the steps relating to PCR anddetection that can be performed in an exemplary microfluidic cartridge.

FIG. 44 is a schematic of an exemplary real-time PCR assay based on theTaqMan® assay.

FIG. 45 is a schematic of positive internal control plasmids which canbe employed.

FIG. 46 depicts the mixing of two fluids (“A”—blue and “B”—orange).

FIGS. 47A and 47B depict a thermally actuated pump 500 based on a phasetransition material (PTM) 510, in a closed (FIG. 47A) and open (FIG.47B) configurations.

FIGS. 47C and 47D show another example of a pump 501 with expancelpolymer 511 in chamber 513 which can be actuated to operate gate 515.

FIG. 48 depicts components of an integrated single, microfluidictechnology based, disposable cartridge.

FIG. 49 depicts DNA capture beads which can be employed.

FIGS. 50A-50J highlight various elements of the microfluidic cartridgeshown in FIGS. 15A and 15B.

FIG. 51 is a side view of a lever assembly 1200, with lever 1210, gearunit 1212, and force member 1214.

FIG. 52 shows a side view of lever assembly 1200, with a microfluidiccartridge in a receiving bay.

FIG. 53 shows a close-up of a receiving bay.

FIG. 54 shows a close-up of the interface between a microfluidiccartridge, thermally conductive, mechanically compliant layer, and athermal stage.

FIG. 55 shows a top view of assembly 1200.

FIG. 56 is a close-up of FIG. 55.

FIGS. 57-59 are a series of pictures of lever 1210 in action. Shown inaddition in gear assembly 1212 can be cam 1228, which enables lever 1210to apply force to plate 1230 coupled to force members 1214.

FIGS. 60 and 61 show views of a microfluidic cartridge withself-piercable reservoirs 1228 and mechanical members 1230 for actuatingthe self piercing reservoirs.

FIGS. 62 and 63 show elements of optical detector elements 1220including light sources 1232 (for example, light emitting diodes),lenses 1234, light detectors 1236 (for example, photodiodes) and filters1238.

FIG. 64 illustrates a microfluidic device.

FIG. 65 is a cross-section of the microfluidic device of FIG. 64 takenalong 5.

FIG. 66 illustrates the retention of herring sperm DNA.

FIG. 67 illustrates the retention and release of DNA from group Bstreptococci;

FIG. 68 illustrates the PCR response of a sample from which inhibitorshad been removed and of a sample from which inhibitors had not beenremoved.

FIG. 69 illustrates the PCR response of a sample prepared in accord withthe technology described herein and a sample prepared using a commercialDNA extraction method.

FIG. 70A illustrates a flow chart showing steps performed during amethod for separating polynucleotides and inhibitors.

FIG. 70B illustrates DNA from samples subjected to the method of FIG.26A.

FIG. 71 is a flow chart that outlines an exemplary set of criteria whichmay be used by the decision algorithm in apparatus 800 to interpret theresults, as described in Example 13.

DETAILED DESCRIPTION

A system, microfluidic cartridge, kit, methods, and computer programproduct, are now further described

Analysis of biological samples often includes determining whether one ormore polynucleotides (e.g., a DNA, RNA, mRNA, or rRNA) can be present inthe sample. For example, one may analyze a sample to determine whether apolynucleotide indicative of the presence of a particular pathogen (suchas a bacterium or a virus) can be present. The polynucleotide may be asample of genomic DNA, or may be a sample of mitochondrial DNA.Typically, biological samples can be complex mixtures. For example, asample may be provided as a blood sample, a tissue sample (e.g., a swabof, for example, nasal, buccal, anal, or vaginal tissue), a biopsyaspirate, a lysate, as fungi, or as bacteria. Polynucleotides to bedetermined may be contained within particles (e.g., cells (e.g., whiteblood cells and/or red blood cells), tissue fragments, bacteria (e.g.,gram positive bacteria and/or gram negative bacteria), fungi, spores).One or more liquids (e.g., water, a buffer, blood, blood plasma, saliva,urine, spinal fluid, or organic solvent) can typically be part of thesample and/or can be added to the sample during a processing step.

Methods for analyzing biological samples include providing a biologicalsample (e.g., a swab), releasing polynucleotides from particles (e.g.,cells such as bacteria) of the sample, amplifying one or more of thereleased polynucleotides (e.g., by polymerase chain reaction (PCR)), anddetermining the presence (or absence) of the amplified polynucleotide(s)(e.g., by fluorescence detection). Biological samples, however,typically include inhibitors (e.g., mucosal compounds, hemoglobin,faecal compounds, and DNA binding proteins) that can inhibit determiningthe presence of polynucleotides in the sample. For example, suchinhibitors can reduce the amplification efficiency of polynucleotides byPCR and other enzymatic techniques for determining the presence ofpolynucleotides. If the concentration of inhibitors is not reducedrelative to the polynucleotides to be determined, the analysis canproduce false negative results. The methods and related systems hereinfor processing biological samples (e.g., samples having one or morepolynucleotides to be determined) are typically able to reduce theconcentration of inhibitors relative to the concentration ofpolynucleotides to be determined by methods further described herein.

Various aspects of a system and a microfluidic cartridge are describedherein.

Additional disclosures of various components thereof may be found inU.S. application Ser. No. 11/580,267, and in provisional applicationSer. No. 60/859,284, the specifications of which are hereby incorporatedby reference.

System Overview

A schematic overview of a system 981 for carrying out analyses describedherein is shown in FIG. 1. The geometric arrangement of the componentsof system 981 shown in FIG. 1 is exemplary and not intended to belimiting. A processor 980, such as a microprocessor, is configured tocontrol functions of various components of the system as shown, and isthereby in communication with each such component. In particular,processor 980 is configured to receive data about a sample to beanalyzed, e.g., from a sample reader 990, which may be a barcode reader,an optical character reader, or an RFID scanner (radio frequency tagreader). For example, the sample identifier can be a handheld bar codereader. Processor 980 is configured to accept user instructions from aninput 984, where such instructions may include instructions to startanalyzing the sample, and choices of operating conditions. Processor 980is also configured to communicate with a display 982, so that, forexample, results of analysis are transmitted to the display.Additionally, processor 980 may transmit one or more questions to bedisplayed on display 982 that prompt a user to provide input in responsethereto. Thus, in certain embodiments, input 984 and display 982 areintegrated with one another. Processor 986 is optionally furtherconfigured to transmit results of an analysis to an output device suchas a printer, a visual display, or a speaker, or a combination thereof.Processor 980 is still further optionally connected via a communicationinterface such as a network interface to a computer network 988. Thecommunication interface can be one or more interfaces selected from thegroup consisting of: a serial connection, a parallel connection, awireless network connection and a wired network connection. Thereby,when the system is suitably addressed on the network, a remote user mayaccess the processor and transmit instructions, input data, or retrievedata, such as may be stored in a memory (not shown) associated with theprocessor, or on some other computer-readable medium that is incommunication with the processor.

Although not shown in FIG. 1, in various embodiments, input 984 caninclude one or more input devices selected from the group consisting of:a keyboard, a touch-sensitive surface, a microphone, a track-pad, aretinal scanner, and a mouse.

Additionally, in various embodiments, the apparatus can further comprisea data storage medium configured to receive data from one or more of theprocessor, an input device, and a communication interface, the datastorage medium being one or more media selected from the groupconsisting of: a hard disk drive, an optical disk drive, a flash card,and a CD-Rom.

Processor 980 is further configured to control various aspects of samplediagnosis, as follows in overview, and as further described in detailherein. The system is configured to operate in conjunction with acomplementary cartridge 994, such as a microfluidic cartridge. Thecartridge is itself configured, as further described herein, to receivea biological sample 996 in a form suitable for work-up and diagnosticanalysis. The cartridge is received by a receiving bay 992 in thesystem. The receiving bay is in communication with a heater 998 thatitself is controlled by processor 980 in such a way that specificregions of the cartridge are heated at specific times during samplework-up and analysis. The processor is also configured to control adetector 999 that receives an indication of a diagnosis from thecartridge 994. The diagnosis can be transmitted to the output device 986and/or the display 982, as described hereinabove.

A suitable processor 980 can be designed and manufactured according to,respectively, design principles and semiconductor processing methodsknown in the art.

The system shown in outline in FIG. 1, as with other exemplaryembodiments described herein, is advantageous because it does notrequire locations within the system suitably configured for storage ofreagents. Neither does the system, or other exemplary embodimentsherein, require inlet or outlet ports that are configured to receivereagents from, e.g., externally stored containers such as bottles,canisters, or reservoirs. Therefore, the system in FIG. 1 isself-contained and operates in conjunction with a microfluidiccartridge, wherein the cartridge has locations within it dedicated toreagent storage.

The system of FIG. 1 may be configured to carry out operation in asingle location, such as a laboratory setting, or may be portable sothat it can accompany, e.g., a physician, or other healthcareprofessional, who may visit patients at different locations. The systemis typically provided with a power-cord so that it can accept AC powerfrom a mains supply or generator. An optional transformer (not shown)built into the system, or situated externally between a power socket andthe system, transforms AC input power into a DC output for use by thesystem. The system may also be configured to operate by using one ormore batteries and therefore is also typically equipped with a batteryrecharging system, and various warning devices that alert a user ifbattery power is becoming too low to reliably initiate or complete adiagnostic analysis.

The system of FIG. 1 may further be configured, in other embodiments,for multiplexed sample analysis. In one such configuration, multipleinstances of a system, as outlined in FIG. 1, are operated inconjunction with one another to accept and to process multiplecartridges, where each cartridge has been loaded with a differentsample. Each component shown in FIG. 1 may therefore be present as manytimes as there are samples, though the various components may beconfigured in a common housing.

In still another configuration, a system is configured to accept and toprocess multiple cartridges, but one or more components in FIG. 1 iscommon to multiple cartridges. For example, a single device may beconfigured with multiple cartridge receiving bays, but a commonprocessor and user interface suitably configured to permit concurrent,consecutive, or simultaneous, control of the various cartridges. It isfurther possible that such an embodiment, also utilizes a single samplereader, and a single output device.

In still another configuration, a system as shown in FIG. 1 isconfigured to accept a single cartridge, but wherein the singlecartridge is configured to process more than 1, for example, 2, 3, 4, 5,or 6, samples in parallel, and independently of one another. Exemplarytechnology for creating cartridges that can handle multiple samples isdescribed elsewhere, e.g., in U.S. application Ser. No. 60/859,284,incorporated herein by reference.

It is further consistent with the present technology that a cartridgecan be tagged, e.g., with a molecular bar-code indicative of the sample,to facilitate sample tracking, and to minimize risk of sample mix-up.Methods for such tagging are described elsewhere, e.g., in U.S. patentapplication Ser. No. 10/360,854, incorporated herein by reference.

Exemplary Systems

FIGS. 2A-2E show exterior perspective views of various configurations ofan exemplary system, as further described herein. FIG. 2A shows aperspective view of a system 2000 for receiving microfluidic cartridge(not shown), and for causing and controlling various processingoperations to be performed a sample introduced into the cartridge. Theelements of system 2000 are not limited to those explicitly shown. Forexample, although not shown, system 2000 may be connected to a hand-heldbar-code reader, as further described herein.

System 2000 comprises a housing 2002, which can be made of metal, or ahardened plastic. The form of the housing shown in FIG. 2A embodiesstylistic as well as functional features. Other embodiments of theinvention may appear somewhat differently, in their arrangement of thecomponents, as well as their overall appearance, in terms of smoothnessof lines, and of exterior finish, and texture. System 2000 furthercomprises one or more stabilizing members 2004. Shown in FIG. 2A is astabilizing foot, of which several are normally present, located atvarious regions of the underside of system 2000 so as to provide balanceand support. For example, there may be three, four, five, six, or eightsuch stabilizing feet. The feet may be moulded into and made of the samematerial as housing 2002, or may be made of one or more separatematerials and attached to the underside of system 2000. For example, thefeet may comprise a rubber that makes it hard for system 2000 to slip ona surface on which it is situated, and also protects the surface fromscratches. The stabilizing member of members may take other forms thanfeet, for example, rails, runners, or one or more pads.

System 2000 further comprises a display 2006, which may be a liquidcrystal display, such as active matrix, an OLED, or some other suitableform. It may present images and other information in color or in blackand white. Display 2006 may also be a touch-sensitive display andtherefore may be configured to accept input from a user in response tovarious displayed prompts. Display 2006 may have an anti-reflectivecoating on it to reduce glare and reflections, from overhead lights inan laboratory setting. Display 2006 may also be illuminated from, e.g.,a back-light, to facilitate easier viewing in a dark laboratory.

System 2000, as shown in FIG. 2A, also comprises a moveable lid 202,having a handle 2008. The lid 2010 can slide back and forward. In FIG.2A, the lid is in a forward position, whereby it is “closed”. In FIG.2B, the lid is shown in a back position, wherein the lid is “open” andreveals a receiving bay 2014 that is configured to receive amicrofluidic cartridge. Of course, as one of ordinary skill in the artwould appreciate, the technology described herein is not limited to alid that slides, or one that slides back and forward. Side to sidemovement is also possible, as is a configuration where the lid is “open”when positioned forward in the device. It is also possible that the lidis a hinged lid, or one that is totally removable.

Handle 2008 performs a role of permitting a user to move lid 2010 formone position to another, and also performs a role of causing pressure tobe forced down on the lid, when in a closed position, so that pressurecan be applied to a cartridge in the receiving bay 2014. In FIG. 2C,handle 2008 is shown in a depressed position, wherein force is therebyapplied to lid 2014, and thus pressure is applied to a cartridgereceived in the receiving bay beneath the lid.

In one embodiment, the handle and lid assembly are also fitted with amechanical sensor that does not permit the handle to be depressed whenthere is no cartridge in the receiving bay. In another embodiment, thehandle and lid assembly are fitted with a mechanical latch that does notpermit the handle to be raised when an analysis is in progress.

A further configuration of system 2000 is shown in FIG. 2D, wherein adoor 2012 is in an open position. Door 2012 is shown in a closedposition in FIGS. 2A-C. The door is an optional component that permits auser to access a heater module 2020, and also a computer-readable mediuminput tray 2022. System 2000 can function without a door that coversheater module 2020 and medium input 2022, but such a door hasconvenience attached to it. Although the door 2012 is shown hinged atthe bottom, it may also be hinged at one of its sides, or at its upperedge. Door 2012 may alternatively be a removable cover, instead of beinghinged. Door 2012, may also be situated at the rear, or side of system2000 for example, if access to the heater module and/or computerreadable medium input is desired on a different face of the system. Itis also consistent with the system herein that the heater module, andthe computer readable medium input are accessed by separate doors on thesame or different sides of the device, and wherein such separate doorsmay be independently hinged or removable.

Heater module 2020 is preferably removable, and is further describedhereinbelow.

Computer readable medium input 2022 may accept one or more of a varietyof media. Shown in FIG. 2D is an exemplary form of input 2022, a CD-Romtray for accepting a CD, DVD, or mini-CD, or mini-DVD, in any of thecommonly used readable, read-writable, and writable formats. Alsoconsistent with the description herein is an input that can acceptanother form of medium, such as a floppy disc, flash memory such asmemory stick, compact flash, smart data-card, or secure-data card, apen-drive, portable USB-drive, zip-disk, and others. Such an input canalso be configured to accept several different forms of media. Such aninput 2022 is in communication with a processor (as described inconnection with FIG. 1, though not shown in FIGS. 2A-E), that can readdata from a computer-readable medium when properly inserted into theinput.

FIG. 2E shows a plan view of a rear of system 2000. Shown are an airvent 2024, or letting surplus heat escape during an analysis. Typically,on the inside of system 2000, and by air vent 2024 and not shown in FIG.2E, is a fan. Other ports shown in FIG. 2E are as follows: a powersocket 2026 for accepting a power cord that will connect system 2000 toa supply of electricity; an ethernet connection 2028 for linking system2000 to a computer network such as a local area network; an phone-jackconnection 2032 for linking system 2000 to a communication network suchas a telephone network; one or more USB ports 2030, for connectingsystem 2000 to one or more peripheral devices such as a printer, or acomputer hard drive; an infra-red port for communicating with, e.g., aremote controller (not shown), to permit a user to control the systemwithout using a touch-screen interface. For example, a user couldremotely issue scheduling commands to system 2000 to cause it to startan analysis at a specific time in the future.

Features shown on the rear of system 2000 may be arranged in anydifferent manner, depending upon an internal configuration of variouscomponents. Additionally, features shown as being on the rear of system2000, may be optionally presented on another face of system 2000,depending on design preference. Shown in FIG. 2E are exemplaryconnections. It would be understood that various other features,including inputs, outputs, sockets, and connections, may be present onthe rear face of system 2000, though not shown, or on other faces ofsystem 2000.

An exploded view of an exemplary embodiment of the apparatus is shown inFIG. 3, particularly showing internal features of apparatus 2000.Apparatus 2000 can comprise a computer readable medium configured withhardware/firmware that can be employed to drive and monitor theoperations on a cartridge used therewith, as well as software tointerpret, communicate and store the results of a diagnostic testperformed on a sample processed in the cartridge. Referring to FIG. 3,typical components of the apparatus 2000 are shown and include, forexample, control electronics 2005, removable heater/sensor module 2020,detector 2009 such as a fluorescent detection module, display screen oroptionally combined display and user interface 2006 (e.g., a medicalgrade touch sensitive liquid crystal display (LCD)). In someembodiments, lid 2010, detector 2009, and handle 2008 can becollectively referred to as slider module 2007. Additional components ofapparatus 2000 may include one or more mechanical fixtures such as frame2019 to hold the various modules (e.g., the heater/sensor module 2020,and/or the slider module 2007) in alignment, and for providingstructural rigidity. Detector module 2009 can be placed in rails tofacilitate opening and placement of cartridge 2060 in the apparatus2000, and to facilitate alignment of the optics upon closing.Heater/sensor module 2020 can be also placed on rails for easy removaland insertion of the assembly.

Embodiments of apparatus 2000 also include software (e.g., forinterfacing with users, conducting analysis and/or analyzing testresults), firmware (e.g., for controlling the hardware during tests onthe cartridge 812), and one or more peripheral communication interfacesshown collectively as 2031 for peripherals (e.g., communication portssuch as USB/Serial/Ethernet to connect to storage such as compact discor hard disk, to connect input devices such as a bar code reader and/ora keyboard, to connect to other computers or storage via a network, andthe like).

Control electronics 840, shown schematically in the block diagram inFIG. 4, can include one or more functions in various embodiments, forexample for, main control 900, multiplexing 902, display control 904,detector control 906, and the like. The main control function may serveas the hub of control electronics 840 in apparatus 2000 and can managecommunication and control of the various electronic functions. The maincontrol function can also support electrical and communicationsinterface 908 with a user or an output device such as a printer 920, aswell as optional diagnostic and safety functions. In conjunction withmain control function 900, multiplexer function 902 can control sensordata 914 and output current 916 to help control heater/sensor module2020. The display control function 904 can control output to and, ifapplicable, interpret input from touch screen LCD 846, which can therebyprovide a graphical interface to the user in certain embodiments. Thedetector function 906 can be implemented in control electronics 840using typical control and processing circuitry to collect, digitize,filter, and/or transmit the data from a detector 2009 such as one ormore fluorescence detection modules.

In various embodiments, fluorescent detection module 2009 can be aminiaturized, highly sensitive fluorescence detection system which can,for example, be capable of real-time analysis of a fluorescent signalemanating from a suitably positioned microfluidic cartridge, as shown inFIG. 5. Detection module 2009 can employ one or more light sources 2850(e.g., light emitting diodes (LED's)), one or more detectors 2852 (e.g.,photodiodes), and one or more filters 2851 and/or lenses 2853. In someembodiments, detection module 2009 can contain multiple (e.g., six)detection elements, where each element can detect one or morefluorescent probes.

In various embodiments, slider module 2007 of the apparatus 2000 canhouse the detection module 2009 (e.g., optical detection system) as wellas mechanical assembly/optics jig 856 to press down on microfluidiccartridge 2020 when the handle 2008 of the slider module 2007 is presseddown. FIGS. 6A and 6B depict the location, in various embodiments, ofthe microfluidic cartridge 2060 after insertion into the apparatus 2000.Optics jig 856 can be suspended from the case of slider module 2007 atone or more (e.g., 4) points. Upon closing slider module 2007 andturning handle 2008 of the apparatus 2000 down, one or more mechanicalactuators 858 (e.g., four cams) can push down plate 860 against one ormore (e.g., 4) springs 862. Upon compression, springs 862 can deliverforce on detector module 2009. A bottom surface 864 of detector module2009 can be made flat (e.g., within 250 microns, typically within 100microns, more typically within 25 microns), and surface 864 can pressupon cartridge 2060, which can have a compliant layer 868 (e.g., Shorehardness approximately 50-70) with a thickness from 0.1-2.5 mm at nocompression, typically about 1.5 mm thick at no compression.Consequently, compression of cartridge 2060, in combination with flatsurface 864, can make the pressure, and thus the thermal contact, moreor less uniform over microfluidic cartridge 2060. One or more springs862 in slider module 2007 can deliver a force (e.g., from 5-500 N,typically about 200-250 N) to generate a pressure (e.g., 2 psi) over thebottom of microfluidic cartridge 2060. FIG. 6B also shows that, when theslider module 2007 can be closed, mechanical features 863 of the slidermodule 2007 can press down on self-pierceable reservoirs 866 of themicrofluidic cartridge 2060, causing the reservoir contents (e.g., DIwater, PCR reagents) be released.

Removable Heater Module

An exemplary removable heater module 2020 is shown in FIG. 7. The moduleis configured to deliver localized heat to various selected regions of acartridge received in the receiving bay 2014. Shown in FIG. 7 is aheater module having a recessed surface 2044 that provides a platformfor supporting a cartridge when in receiving bay 2014. In oneembodiment, the cartridge rests directly on surface 2044. Surface 2044is shown as recessed, in FIG. 7, but need not be so.

Area 2044 is configured to accept a microfluidic cartridge in a singleorientation. Therefore area 2044 can be equipped with a registrationmember such as a mechanical key that prevents a user from placing acartridge into receiving bay 2014 in the wrong configuration. Shown inFIG. 7 as an exemplary mechanical key 2045 is a diagonally cutout cornerof area 2044 into which a complementarily cutout corner of amicrofluidic cartridge fits (see, e.g., FIG. 9). Other registrationmembers are consistent with the apparatus described herein: for example,a feature engineered on one or more edges of a cartridge including butnot limited to: several, such as two or more, cut-out corners, one ormore notches cut into one or more edges of cartridge of FIG. 9; or oneor more protrusions fabricated into one or more edges of cartridge ofFIG. 9. Alternative registration members include one or more lugs orbumps engineered into an underside of a cartridge, complementary to oneor more recessed sockets or holes in surface 2044. Alternativeregistration members include one or more recessed sockets or holesengineered into an underside of a cartridge, complementary to one ormore lugs or bumps on surface 2044. In general, the pattern of featuresis such that the cartridge possesses at least one element of asymmetryso that it can only be inserted in a single orientation into thereceiving bay.

Also shown in FIG. 7 is a hand-grasp 2042 that facilitates removal andinsertion of the heater module by a user. Cutaway 2048 permits a user toeasily remove a cartridge from receiving bay 2014 after a processing runwhere, e.g., a user's thumb of finger when grabbing the top of thecartridge, is afforded comfort space by cutaway 2048. Both cutaways 2042and 2048 are shown as semicircular recesses in the embodiment of FIG. 7,but it would be understood that they are not so limited in shape. Thus,rectangular, square, triangular, oval, and other shaped recesses arealso consistent with a heater module as described herein.

In the embodiment of FIG. 7, which is designed to be compatible with thesystem of FIGS. 2A-E, the front of the heater module is at the left ofthe figure. At the rear of heater module 2020 is an electricalconnection 2050, such as an RS-232 connection, that permits electricalsignals to be directed to heaters located at specific regions of area2044 during sample processing and analysis, as further described herein.Thus, underneath area 2044 and not shown in FIG. 7 can be an array ofheat sources, such as resistive heaters, that are configured to alignwith specified locations of a microfluidic cartridge properly insertedinto the receiving bay. Surface 2044 is able to be cleaned periodicallyto ensure that any liquid spills that may occur during sample handlingdo not cause any short circuiting.

Other non-essential features of heater module 2020 are as follows. Oneor more air vents 2052 can be situated on one or more sides (such asfront, rear, or flanking) or faces (such as top or bottom) of heatermodule 2020, to permit excess heat to escape, when heaters underneathreceiving bay 2014, are in operation. The configuration of air vents inFIG. 7 is exemplary and it would be understood that other numbers andshapes thereof are consistent with routine fabrication and use of aheater module. For example, although 5 square air vents are shown, othernumbers such as 1, 2, 3, 4, 6, 8, or 10 air vents are possible, arrangedon one side, or spread over two or more sides and/or faces of the heatermodule. In further embodiments, air vents may be circular, rectangular,oval, triangular, polygonal, and having curved or squared vertices, orstill other shapes, including irregular shapes.

Heater module 2020 may further comprise one or more guiding members 2047that facilitate inserting the heater module into an apparatus as furtherdescribed herein for an embodiment in which heater module 2020 isremovable by a user. Heater module is advantageously removable becauseit permits system 2000 to be easily reconfigured for a different type ofanalysis, such as employing a different cartridge with a differentregistration member and/or microfluidic network, in conjunction with thesame or a different sequence of processing operations. In otherembodiments, heater module 2020 is designed to be fixed and onlyremovable, e.g., for cleaning, replacement, or maintenance, by themanufacturer or an authorized maintenance agent, and not routinely bythe user. Guiding members may perform one or more roles of ensuring thatthe heater module is aligned correctly in the apparatus, and ensuringthat the heater module makes a tight fit and does not significantly moveduring processing and analysis of a sample, or during transport of theapparatus. Guiding members shown in the embodiment of FIG. 7 are oneither side of receiving bay 2044 and stretch along a substantialfraction of the length of module 2020. Other guiding members areconsistent with use herein, and include but are not limited to othernumbers of guiding members such as 1, 3, 4, 5, 6, or 8, and otherpositions thereof, including positioned in area 2051 of module 2020.Guiding members 2047 are shown having a non-constant thickness alongtheir lengths. It is consistent herein that other guiding members mayhave essentially constant thickness along their lengths.

Adjacent receiving bay 2014 is a non-contact heating element 2046, suchas lamp, set into a recessed area. 2053. Recessed area 2053 may also beconfigured with a reflector, or a reflective coating, so that as much asthermal and optical energy from non-contact heating element 2046 aspossible is directed outwards towards receiving bay 2014. Element 2046is a heat lamp in certain embodiments. Element 2046 is configured toreceive electrical energy and thereby heat up from the effects ofelectrical resistance. Element 2046 provides a way of heating a raisedregion of a cartridge received in receiving bay 2014. The raised regionof the cartridge (see, e.g., FIG. 9) may contain a lysis chamber, andapplication of heat from non-contact heating element 2046 can have theeffect of lysing cells within the lysis chamber.

Also shown in FIG. 7 is an optional region of fluorescent material, suchas optically fluorescent material, 2049 on area 2051 of heater module2020. The region of fluorescent material is configured to be detected bya detection system further described herein. The region 2049 is used forverifying the state of optics in the detection system prior to sampleprocessing and analysis and therefore acts as a control, or a standard.For example, in one embodiment a lid of the apparatus (see, e.g., FIG.2A) when in an open position permits ambient light to reach region 2049and thereby cause the fluorescent material to emit a characteristicfrequency or spectrum of light that can be measured by the detector for,e.g., standardization or calibration purposes. In another embodiment,instead of relying on ambient light to cause the fluorescent material tofluoresce, light source from the detection system itself, such as one ormore LED's, is used. The region 2049 is therefore positioned to alignwith a position of a detector. Region 2049 is shown as rectangular, butmay be configured in other shapes such as square, circular, elliptical,triangular, polygonal, and having curved or squared vertices. It is alsoto be understood that the region 2049 may be situated at other places onthe heater module 2020, according to convenience and in order to becomplementary to the detection system deployed.

Heater module 2020 also comprises an array of heaters, situated beneatharea 2044 and not shown in FIG. 7. As further described herein, suchheaters may be resistive heaters, configured to heat specifically and atspecific times, according to electrical signals received.

In particular and not shown in FIG. 7, heater/sensor module 2020 caninclude, for example, a multiplexing function 902 in a discretemultiplexing circuit board (MUX board), one or more heaters (e.g., amicroheater), one or more temperature sensors (optionally combinedtogether as a single heater/sensor unit with one or more respectivemicroheaters, e.g., as photolithographically fabricated on fused silicasubstrates), and a non-contact heating element 2046. The micro-heatersand non-contact heating element can provide thermal energy that canactuate various microfluidic components on a suitably positionedmicrofluidic cartridge. A sensor (e.g., as a resistive temperaturedetector (RTD)) can enable real time monitoring of the micro-heaters andthe non-contact heater(s) 2046, for example through a feedback basedmechanism to allow for control of the temperature. One or moremicroheaters can be aligned with corresponding microfluidic components(e.g., valves, pumps, gates, reaction chambers) to be heated on asuitably positioned microfluidic cartridge. A microheater can bedesigned to be slightly bigger than the corresponding microfluidiccomponent(s) on the microfluidic cartridge so that even though thecartridge may be slightly misaligned, such as off-centered, from theheater, the individual components can be heated effectively.

Non-contact heater 2046 can also serve as a radiation heat source toheat one section of a suitably positioned microfluidic cartridge. Forexample, a 20 W Xenon lamp may be used as the non-contact heatingelement 2046. In various embodiments, heater/sensor module 2020 can bespecific to particular cartridge designs and can be easily replaceablethrough the front panel of the apparatus 800. Heater/sensor module 2020can be configured to permit cleaning of heating surface 2044 with commoncleaning agents (e.g., a 10% bleach solution).

Referring to FIGS. 8A and 8B, an exemplary set of heaters configured toheat, cyclically, PCR reaction zone 1001 is shown. It is to beunderstood that heater configurations to actuate other regions of amicrofluidic cartridge such as other gates, valves, and actuators, maybe designed and deployed according to similar principles to thosegoverning the heaters shown in FIGS. 8A and 8B. An exemplary PCRreaction zone 1001, typically a chamber or channel having a volume ˜1.6μl, is configured with a long side and a short side, each with anassociated heating element. A PCR reaction zone may also be referred toas a PCR reactor, herein. The apparatus therefore preferably includesfour heaters disposed along the sides of, and configured to heat, agiven PCR reaction zone, as shown in the exemplary embodiment of FIG.8A: long top heater 1005, long bottom heater 1003, short left heater1007, and short right heater 1009. The small gap between long top heater1005 and long bottom heater 1003 results in a negligible temperaturegradient (less than 1° C. across the width of the PCR channel at anypoint along the length of the PCR reaction zone) and therefore aneffectively uniform temperature throughout the PCR reaction zone. Theheaters on the short edges of the PCR reactor provide heat to counteractthe gradient created by the two long heaters from the center of thereactor to the edge of the reactor.

It would be understood by one of ordinary skill in the art that stillother configurations of one or more heater(s) situated about a PCRreaction zone are consistent with the methods and apparatus describedherein. For example, a ‘long’ side of the reaction zone can beconfigured to be heated by two or more heaters. Specific orientationsand configurations of heaters are used to create uniform zones ofheating even on substrates having poor thermal conductivity because thepoor thermal conductivity of glass, or quartz, or fused silicasubstrates is utilized to help in the independent operation of variousmicrofluidic components such as valves and independent operation of thevarious PCR lanes. It would be further understood by one of ordinaryskill in the art, that the principles underlying the configuration ofheaters around a PCR reaction zone are similarly applicable to thearrangement of heaters adjacent to other components of the microfluidiccartridge, such as actuators, valves, and gates.

In certain embodiments, each heater has an associated temperaturesensor. In the embodiment of FIG. 8A, a single temperature sensor 1011is used for both long heaters. A temperature sensor 1013 for short leftheater, and a temperature sensor 1015 for short right heater are alsoshown. The temperature sensor in the middle of the reactor is used toprovide feedback and control the amount of power supplied to the twolong heaters, whereas each of the short heaters has a dedicatedtemperature sensor placed adjacent to it in order to control it.Temperature sensors are preferably configured to transmit informationabout temperature in their vicinity to the processor at such times asthe heaters are not receiving current that causes them to heat. This canbe achieved with appropriate control of current cycles.

In order to reduce the number of sensor or heater elements required tocontrol a PCR heater, we may use the heaters to sense as well as heat,and thereby obviate the need to have a separate dedicated sensor foreach heater. In another embodiment, each of the four heaters may bedesigned to have an appropriate wattage, and connect the four heaters inseries or in parallel to reduce the number ofelectronically-controllable elements from 4 to just 1, thereby reducingthe burden on the associated electronic circuitry.

FIG. 8B shows expanded views of heaters and temperature sensors used inconjunction with a PCR reaction zone of FIG. 8A. Temperature sensors1001 and 1013 are designed to have a room temperature resistance ofapproximately 200-300 ohms. This value of resistance is determined bycontrolling the thickness of the metal layer deposited (e.g., a sandwichof 400 Å TiW/3,000 Å Au/400 Å TiW), and etching the winding metal lineto have a width of approximately 10-25 μm and 20-40 mm length. The useof metal in this layer gives it a temperature coefficient of resistivityof the order of 0.5-20° C./ohms, preferably in the range of 1.5-3°C./ohms. Measuring the resistance at higher temperatures enablesdetermination of the exact temperature of the location of these sensors.

The configuration for uniform heating, shown in FIG. 8A for a single PCRreaction zone, can also be applied to a multi-lane PCR cartridge inwhich multiple independent PCR reactions occur.

Each heater can be independently controlled by a processor and/orcontrol circuitry used in conjunction with the apparatus describedherein. FIG. 8C shows thermal images, from the top surface of amicrofluidic cartridge when heated by heaters configured as in FIGS. 8Aand 8B, when each heater in turn is activated, as follows: (A): Long Toponly; (B) Long Bottom only; (C) Short Left only; (D) Short Right only;and (E) All Four Heaters on. Panel (F) shows a view of the reaction zoneand heaters on the same scale as the other image panels in FIG. 8C. Alsoshown in the figure is a temperature bar.

Microfluidic Cartridge

FIG. 9 shows a perspective view of an exterior of an exemplarymicrofluidic cartridge 2060 for use in conjunction with the systemdescribed herein. Present in cartridge 2060 is at least one reagentpackage 2062. Four such reagent packages are shown, though other numbersof such packages, such as but not limited to one, two, three, five, six,eight, ten, and twelve, are possible, depending upon application.Cartridge 2060 further comprises a tower 2064 that contains reagents,and is fitted with an inlet 2066, such as a luer, through which aportion of a biological sample can be introduced. Tower 2064 can alsocomprise one or more chambers such as a bulk lysis chamber 2065 and awaste chamber 2067. Waste chamber 2067 may have a vent 2069 forreleasing gases such as air.

Other than tower 2064, cartridge 2060 is substantially planar such thatit can be easily handled by an operator and can be easily matched to acomplementary receiving bay of an apparatus such as shown in FIG. 1.

Cartridge 2060 further comprises a port 2068 through which a detectorcan receive a signal directly or indirectly from one or morepolynucleotides in the sample, during processing or amplification, inorder to provide a user with a diagnostic result on the sample.

Cartridge 2060 can further comprise a registration member such as amechanical key, complementary to a corresponding registration member inthe receiving bay. Shown in FIG. 9 is an exemplary registration member2071, a corner cut-out from the cartridge.

The integrated system, as described herein, comprises an apparatusconfigured to receive a microfluidic cartridge, and a microfluidiccartridge. It is consistent with the system described herein that anumber of different configurations of microfluidic cartridge, andpurposes thereof, are compatible with suitably configured apparati.Thus, for example, although benefits are described wherein a singlecartridge is capable of accepting a collected biological sample, workingup the sample, including lysing cells to liberate and collectpolynucleotides contained therein, applying pre-amplificationpreparatory steps to the polynucleotides, amplifying thepolynucleotides, and causing the amplified polynucleotides to bedetected, it is also consistent with the descriptions herein that othermicrofluidic cartridges can be used. Such other cartridges can beconfigured to carry out fewer, such as one or more, of theaforementioned steps, and, correspondingly the apparatus for usetherewith is configured to cause fewer such steps to be effectuated. Itis to be understood therefore, that when presenting various exemplaryconfigurations of microfluidic cartridge herein, the various componentsthereof can be used interchangeably (e.g., an exemplary valve describedin connection with one cartridge can also be used in a network describedin connection with another cartridge) both without modification, andwith suitable adjustments or modifications of geometry or size, asappropriate.

Aspects of Microfluidic Cartridges

Accordingly, the technology herein also comprises a microfluidiccartridge having attributes, as follows. Thus the technology includes amicrofluidic cartridge that is configured to process one or morepolynucleotides, e.g., to concentrate the polynucleotide(s) and/or toseparate the polynucleotide(s) from inhibitor compounds, (e.g.,hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds,DNA binding proteins, or a saccharide) that might inhibit detectionand/or amplification of the polynucleotides.

The microfluidic cartridge can be configured to contact thepolynucleotides and a relatively immobilized compound thatpreferentially associates with (e.g., retains) the polynucleotides asopposed to inhibitors. An exemplary compound is a poly-cationicpolyamide (e.g., poly-L-lysine and/or poly-D-lysine), orpolyethyleneimine (PEI), which may be bound to a surface (e.g., surfacesof one or more particles). The compound retains the polynucleotides sothat the polynucleotides and inhibitors may be separated, such as bywashing the surface to which the compound and associated polynucleotidesare bound. Upon separation, the association between the polynucleotideand compound may be disrupted to release (e.g., separate) thepolynucleotides from the compound and surface.

In some embodiments, the surface (e.g., surfaces of one or moreparticles) can be modified with a poly-cationic substance such as apolyamide or PEI, which may be covalently bound to the surface. Thepoly-cationic polyamide may include at least one of poly-L-lysine andpoly-D-lysine. In some embodiments, the poly-cationic polyamide (e.g.,the at least one of the poly-L-lysine and the poly-D-lysine) has anaverage molecular weight of at least about 7500 Da. The poly-cationicpolyamide (e.g., the at least one of the poly-L-lysine and thepoly-D-lysine) may have an average molecular weight of less than about35,000 Da (e.g., an average molecular weight of less than about 30,000Da (e.g., an average molecular weight of about 25,000 Da)). Thepoly-cationic polyamide (e.g., the at least one of the poly-L-lysine andthe poly-D-lysine) may have a median molecular weight of at least about15,000 Da. The poly-cationic polyamide (e.g., the at least one of thepoly-L-lysine and the poly-D-lysine) may have a median molecular weightof less than about 25,000 Da (e.g., a median molecular weight of lessthan about 20,000 Da (e.g., a median molecular weight of about 20,000Da). If the polycationic material is PEI, its molecular weight ispreferably in the range 600-800 Daltons.

In other embodiments, the microfluidic cartridge includes a surfacehaving a poly-cationic polyamide or PEI bound thereto and a sampleintroduction passage in communication with the surface for contactingthe surface with a fluidic sample.

In some embodiments, the apparatus includes a heat source configured toheat an aqueous liquid in contact with the surface to at least about 65°C.

In some embodiments, the cartridge includes a reservoir of liquid havinga pH of at least about 10 (e.g., about 10.5 or more). The cartridge canbe configured to contact the surface with the liquid (e.g., by actuatinga pressure source to move the liquid).

Another aspect of the microfluidic cartridge relates to a retentionmember, e.g., a plurality of particles such as beads, comprising boundPEI, or poly-lysine, e.g., poly-L-lysine, and related methods andsystems. An exemplary method for processing a sample includes contactinga retention member with a mixture that includes providing a mixtureincluding a liquid and an amount of polynucleotide. The retention membermay be configured to preferentially retain polynucleotides as comparedto polymerase chain reaction inhibitors. Substantially all of the liquidin the mixture can be removed from the retention member. Thepolynucleotides can be released from the retention member. Thepolynucleotide may have a size of less than about 7.5 Mbp.

The liquid may be a first liquid, and removing substantially all of theliquid from the retention member may include contacting the retentionmember with a second liquid.

Contacting the retention member with a second liquid can includeactuating a thermally actuated pressure source to apply a pressure tothe second liquid. Contacting the retention member with a second liquidcan include opening a thermally actuated valve to place the secondliquid in fluid communication with the retention member.

The second liquid may have a volume of less than about 50 microliters,and may include a detergent (e.g., SDS).

The retention member may include a surface having a compound configuredto bind polynucleotides preferentially to polymerase chain reactioninhibitors (such inhibitors including, for example, hemoglobin,peptides, faecal compounds, humic acids, mucousol compounds, DNA bindingproteins, or a saccharide).

The surface may include a poly-lysine (e.g., poly-L-lysine and/orpoly-D-lysine) or PEI.

Releasing polynucleotides from the retention member may include heatingthe retention member to a temperature of at least about 50° C. (e.g., atabout 65° C.). The temperature may be insufficient to boil the liquid inthe presence of the retention member during heating. The temperature maybe 100° C. or less (e.g., less than 100° C., about 97° C. or less). Thetemperature may be maintained for less than about 10 minutes (e.g., forless than about 5 minutes, for less than about 3 minutes). The releasingmay be performed without centrifugation of the retention member.

In certain embodiments, PCR inhibitors can be rapidly removed fromclinical samples to create a PCR-ready sample. Methods herein thereforemay comprise the preparation of a polynucleotide-containing sample thatcan be substantially free of inhibitors. Such samples may be preparedfrom, e.g., crude lysates resulting from thermal, chemical, ultrasonic,mechanical, electrostatic, and other lysing techniques. The samples maybe prepared without centrifugation. The samples may be prepared usingother microfluidic devices or on a larger scale.

The retention member may be used to prepare polynucleotide samples forfurther processing, such as amplification by polymerase chain reaction.In certain embodiments, more than 90% of a polynucleotide present in asample may be bound to the retention member, released, and recovered.

In certain embodiments, a polynucleotide may be bound to the retentionmember, released, and recovered, in less than about 10 minutes (e.g.,less than about 71/2 minutes, less than about 5 minutes, or less thanabout 3 minutes).

A polynucleotide may be bound to a retention member, released, andrecovered without subjecting the polynucleotide, retention member,and/or inhibitors to centrifugation. Separating the polynucleotides andinhibitors generally excludes subjecting the polynucleotides,inhibitors, processing region, and/or retention member to sedimentation(e.g., centrifugation).

In various embodiments, the microfluidic cartridge can include a PCRreagent mixture comprising a polymerase enzyme and a plurality ofnucleotides. The PCR reagent mixture can be in the form of one or morelyophilized pellets, and the microfluidic network can be configured tocontact the PCR pellet with liquid to create a PCR reagent mixturesolution.

In various embodiments, the microfluidic cartridge can be configured tocouple heat from an external heat source with the PCR reagent mixtureand the neutralized polynucleotide sample under thermal cyclingconditions suitable for creating PCR amplicons from the neutralizedpolynucleotide sample.

In various embodiments, the PCR reagent mixture can further include apositive control plasmid and a fluorogenic hybridization probe selectivefor at least a portion of the plasmid.

In various embodiments, the microfluidic cartridge can include anegative control polynucleotide, wherein the microfluidic network can beconfigured to independently contact each of the neutralizedpolynucleotide sample and the negative control polynucleotide with thePCR reagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide.

In various embodiments, the microfluidic cartridge can include at leastone probe that can be selective for a polynucleotide sequence, whereinthe microfluidic cartridge can be configured to contact the neutralizedpolynucleotide sample or a PCR amplicon thereof with the probe. Theprobe can be a fluorogenic hybridization probe. The fluorogenichybridization probe can include a polynucleotide sequence coupled to afluorescent reporter dye and a fluorescence quencher dye. The PCRreagent mixture can further include a positive control plasmid and aplasmid fluorogenic hybridization probe selective for at least a portionof the plasmid and the microfluidic cartridge can be configured to allowindependent optical detection of the fluorogenic hybridization probe andthe plasmid fluorogenic hybridization probe.

In various embodiments, the probe can be selective for a polynucleotidesequence that can be characteristic of an organism, for example anyorganism that employs deoxyribonucleic acid or ribonucleic acidpolynucleotides. Thus, the probe can be selective for any organism.Suitable organisms include mammals (including humans), birds, reptiles,amphibians, fish, domesticated animals, farmed animals, wild animals,extinct organisms, bacteria, fungi, viruses, plants, and the like. Theprobe can also be selective for components of organisms that employtheir own polynucleotides, for example mitochondria. In someembodiments, the probe can be selective for microorganisms, for example,organisms used in food production (for example, yeasts employed infermented products, molds or bacteria employed in cheeses, and the like)or pathogens (e.g., of humans, domesticated or wild mammals,domesticated or wild birds, and the like). In some embodiments, theprobe can be selective for organisms selected from the group consistingof gram positive bacteria, gram negative bacteria, yeast, fungi,protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that can be characteristic of an organism selected from thegroup consisting of Staphylococcus spp., e.g., S. epidermidis, S.aureus, Methicillin-resistant Staphylococcus aureus (MRSA),Vancomycin-resistant Staphylococcus; Streptococcus (e.g., α, β orγ-hemolytic, Group A, B, C, D or G) such as S. pyogenes, S. agalactiae;E. faecalis, E. durans, and E. faecium (formerly S. faecalis, S. durans,S. faecium); nonenterococcal group D streptococci, e.g., S. bovis and S.equines; Streptococci viridans, e.g., S. mutans, S. sanguis, S.salivarius, S. mitior, A. milleri, S. constellatus, S. intermedius, andS. anginosus; S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides,N. gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E.rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanoviiand L. seeligeri; Bacillus, e.g., B. anthracis, B. cereus, B. subtilis,B. subtilus niger, B. thuringiensis; Nocardia asteroids; Legionella,e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii;Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E.coli, E. coliO157:H7); Klebsiella, Enterobacter, Serratia, Proteus,Morganella, Providencia, Yersinia, and the like, e.g., Salmonella, e.g.,S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii),S. dublin S. choleraesuis, S. enteritidis, S. typhimurium, S.heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S.saint-paul; Shigella, e.g., subgroups: A, B, C, and D, such as S.flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis,P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia(P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y.pestis, Y. enterocolitica; Haemophilus, e.g., H. influenzae, H.parainfluenzae H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus,B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis;Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei,Burkholderia mallei, Burkholderia cepacia and Stenotrophomonasmaltophilia; Campylobacter, e.g., C. fetus fetus, C. jejuni, C. pylori(Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus,V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and thenonagglutinable vibrios; Clostridia, e.g., C. perfringens, C. tetani, C.difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides,e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B.ovatus, B. caccae, and B. merdae; Prevotella, e.g., P. melaninogenica;genus Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum,T. pallidum subspecies pertenue, T. carateum, and T. pallidum subspeciespallidum; genus Borrelia, e.g., B. burgdorferi; genus Leptospira;Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus;Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. aviumM. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, theM. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M.asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M.malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma,e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae,M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M.genitalium; Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T.vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H.capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides,e.g., C. immitis; Blastomyces, e.g. B. dermatitidis; Paracoccidioides,e.g., P. brasiliensis; Penicillium, e.g., P. marneffei; Sporothrix,e.g., S. schenckii; Rhizopus, Rhizomucor, Absidia, and Basidiobolus;diseases caused by Bipolaris, Cladophialophora, Cladosporium,Drechslera, Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis,Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T.beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P.falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa ofthe genus Trypanosoma, e.g., T. cruzi; Leishmania, e.g., L. donovani, L.major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis;Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria orAcanthamoeba; Entamoeba histolytica; Giardia lamblia; genusCryptosporidium, e.g., C. parvum; Isospora belli; Cyclosporacayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostomaduodenale or Necator americanus; Strongyloides stercoralis Toxocara,e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella,e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocercavolvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S.japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium;Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis;Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothriumlatum; Taenia, e.g., T. saginata, T. solium; Echinococcus, e.g., E.granulosus, E. multilocularis; Picornaviruses, rhinoviruses echoviruses,coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3,and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;varicella-zoster virus; human T-lymphotrophic virus (type I and typeII); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae,Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis(alphaviruses e.g., Venezuelan equine encephalitis, eastern equineencephalitis, western equine encephalitis); Viral hemorrhagic fevers(filoviruses, e.g., Ebola, Marburg, and arenaviruses, e.g., Lassa,Machupo); Smallpox (variola); retroviruses e.g., human immunodeficiencyviruses 1 and 2; human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33,and 35.

In various embodiments, the probe can be selective for a polynucleotidesequence that can be characteristic of an organism selected from thegroup consisting of Pseudomonas aeruginosa, Proteus mirabilis,Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli,Acinetobacter Baumannii, Serratia marcescens, Enterobacter aerogenes,Enterococcus faecium, vancomycin-resistant enterococcus (VRE),Staphylococcus aureus, methecillin-resistant Staphylococcus aureus(MRSA), Streptococcus viridans, Listeria monocytogenes, Enterococcusspp., Streptococcus Group B, Streptococcus Group C, Streptococcus GroupG, Streptococcus Group F, Enterococcus faecalis, Streptococcuspneumoniae, Staphylococcus epidermidis, Gardenerella vaginalis,Micrococcus sps., Haemophilus influenzae, Neisseria gonorrhoeee,Moraxella catarrahlis, Salmonella sps., Chlamydia trachomatis,Peptostreptococcus productus, Peptostreptococcus anaerobius,Lactobacillus fermentum, Eubacterium lentum, Candida glabrata, Candidaalbicans, Chlamydia spp., Camplobacter spp., Salmonella spp., smallpox(variola major), Yersina Pestis, Herpes Simplex Virus I (HSV I), andHerpes Simplex Virus II (HSV II).

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of Group B Streptococcus.

The technology herein also comprises a microfluidic cartridge having acomponent for inhibiting motion of fluid. The component comprises achannel, a first mass of a thermally responsive substance (TRS) disposedon a first side of the channel, a second mass of a TRS disposed on asecond side of the channel opposite the first side of the channel, a gaspressure source associated with the first mass of the TRS. Actuation ofthe gas pressure source drives the first mass of the TRS into the secondmass of the TRS and obstructs the channel.

The microfluidic cartridge can include a second gas pressure sourceassociated with the second mass of the TRS. Actuation of the second gaspressure source drives the second mass of TRS into the first mass ofTRS. At least one (e.g., both) of the first and second masses of TRS maybe a wax.

Another aspect of the microfluidic cartridge includes a component forobstructing a channel of a microfluidic cartridge. A mass of a TRS canbe heated and driven across the channel (e.g., by gas pressure) into asecond mass of TRS. The second mass of TRS may also be driven (e.g., bygas pressure) toward the first mass of TRS.

Another aspect of the microfluidic cartridge is an actuator. Theactuator includes a channel, a chamber connected to the channel, atleast one reservoir of encapsulated liquid disposed in the chamber, anda gas surrounding the reservoir within the chamber. Heating the chamberexpands the reservoir of encapsulated liquid and pressurizes the gas.Typically the liquid has a boiling point of about 90° C. or less. Theliquid may be a hydrocarbon having about 10 carbon atoms or fewer. Theliquid may be encapsulated by a polymer.

An actuator may include multiple reservoirs of encapsulated liquiddisposed in the chamber. The multiple reservoirs may be dispersed withina solid (e.g., a wax). The multiple reservoirs may be disposed within aflexible enclosure (e.g., a flexible sack).

Another aspect of the microfluidic cartridge includes pressurizing a gaswithin a chamber of the device to create a gas pressure sufficient tomove a liquid within a channel of the microfluidic device. Pressurizingthe gas typically expands at least one reservoir of encapsulated liquiddisposed within the chamber. Expanding the at least one reservoir caninclude heating the chamber. Pressurizing the gas can include expandingmultiple reservoirs of encapsulated liquid.

Another aspect of a microfluidic cartridge for use herein includescombining (e.g., mixing) first and second liquid volumes. The deviceincludes a mass of a temperature responsive substance (TRS) thatseparates first and second channels of the device. The device can beconfigured to move a first liquid along the first channel so that aportion (e.g., a medial portion) of the first liquid can be adjacent theTRS, and to move a second liquid along the second channel so that aportion (e.g., a medial portion) of second liquid can be adjacent theTRS. A heat source can be actuated to move the TRS (e.g., by melting,dispersing, fragmenting). The medial portions of the first and secondliquids typically combine without being separated by a gas interface.Typically, only a subset of the first liquid and a subset of the secondliquid can be combined. The liquids mix upon being moved along a mixingchannel. The liquids when combined should be moved at least two dropletlengths to get good mixing by interlayering and transverse diffusion(perpendicular to the length of the microchannel) without having to relyon longitudinal diffusion alone for mixing (see, also, e.g.,“Mathematical modeling of drop mixing in a slit-type microchannel”, KHandique, et al., J. Micromech. Microeng., 11 548-554, (2001),incorporated herein by reference). By moving the combined drop by a droplength, the liquid in the middle of the receding drop is caused to moveto the front of the leading drop and then towards the channel wall. Atthe receding end of the drop, liquid moves from the wall towards thecenter of the drop. This motion of the drop results in interlayeringbetween the two liquids. Further interlayering can be achieved byrepeating the method additional times, such as over further droplengths.

The microfluidic cartridge further includes a lyophilized reagentparticle. In some embodiments, the lyophilized particles includemultiple smaller particles each having a plurality of ligands thatpreferentially associate with polynucleotides as compared to PCRinhibitors. The lyophilized particles can also (or alternatively)include lysing reagents (e.g., enzymes) configured to lyse cells torelease polynucleotides. The lyophilized particles can also (oralternatively) include enzymes (e.g., proteases) that degrade proteins.

Cells can be lysed by combining a solution of the cells, e.g., in amicrofluidic droplet, with the lyophilized particles therebyreconstituting the particles. The reconstituted lysing reagents lyse thecells. The polynucleotides associate with ligands of the smaller.particles. During lysis, the solution may be heated (e.g., radiativelyusing a lamp such as a heat lamp, or by a contact heat source.

In some embodiments, lyophilized particles include reagents (e.g.,primers, control plasmids, polymerase enzymes) for performing PCR.

Another aspect of the microfluidic cartridge includes a liquid reservoircapable of holding a liquid (e.g., a solvent, a buffer, a reagent, orcombination thereof). In general, the reservoir can have one or more ofthe following features, as further described in internationalapplication publication no. WO2006/079082.

The reservoir can include a wall that can be manipulated (e.g., pressedor depressed) to decrease a volume within the reservoir. For example,the reservoir can include a piercing member (e.g., a needle-like orotherwise pointed or sharp member) that ruptures another portion of thereservoir (e.g., a portion of the wall) to release liquid. The piercingmember can be internal to the reservoir such that the piercing memberruptures the wall from an inner surface of the reservoir (e.g., wall)outwards.

In general, the wall resists passage of liquid or vapor therethrough. Insome embodiments, the wall lacks stretchiness. The wall may be flexible.The wall may be, e.g., a metallic layer, e.g., a foil layer, a polymer,or a laminate including a combination thereof. The wall may be formed byvacuum formation (e.g., applying a vacuum and heat to a layer ofmaterial to draw the layer against a molding surface). The moldingsurface may be concave such that the wall can be provided with agenerally convex surface.

Exemplary liquids held by the reservoir include water and aqueoussolutions including one or more salts (e.g., magnesium chloride, sodiumchloride, Tris buffer, or combination thereof). The reservoir can retainthe liquid (e.g., without substantial evaporation thereof) for a periodof time (e.g., at least 6 months or at least a year). In someembodiments, less than 10% (e.g., less than about 5%) by weight of theliquid evaporates over a year.

The piercing member may be an integral part of a wall of the reservoir.For example, the reservoir can include a wall having an internalprojection, which may be in contact with liquid in the reservoir. Thereservoir also includes a second wall opposite the piercing member.During actuation, the piercing member can be driven through the secondwall (e.g., from the inside out) to release liquid.

In some embodiments, a maximum amount of liquid retained by a reservoircan be less than about 1 ml. For example, a reservoir may hold about 500microliters or less (e.g., 300 microliters or less). Generally, areservoir holds at least about 25 microliters (e.g., at least about 50microliters). The reservoir can introduce within about 10% of theintended amount of liquid (e.g., 50±5 μl).

The reservoir can deliver a predetermined amount of liquid that can besubstantially air-free (e.g., substantially gas-free). Upon introductionof the liquid, the substantially air and/or gas free liquid produces fewor no bubbles large enough to obstruct movement of the liquid within themicrofluidic device. Use of a piercing member internal to the reservoircan enhance an ability of the reservoir to deliver substantially airand/or gas free liquids.

In some embodiments, the reservoir can be actuated to release liquid bypressing (e.g., by one's finger or thumb or by mechanical pressureactuation). The pressure may be applied directly to a wall of thereservoir or to a plunger having a piercing member. In variousembodiments, minimal pressure can be required to actuate the reservoir.An automated system can be used to actuate (e.g., press upon) aplurality of reservoirs simultaneously or in sequence.

Actuation of the reservoir may include driving a piercing member througha wall of the reservoir. In some embodiments, the reservoir does notinclude a piercing member. Instead, internal pressure generated withinthe reservoir ruptures a wall of the reservoir allowing liquid to enterthe microfluidic device.

Upon actuating a reservoir to introduce liquid into the microfluidicdevice, liquid generally does not withdraw back into the reservoir. Forexample, upon actuation, the volume of the reservoir may decrease tosome minimum but generally does not increase so as to withdraw liquidback into the reservoir. For example, the reservoir may stay collapsedupon actuation. In such embodiments, the flexible wall may be flexiblebut lack hysteresis, elasticity, or stretchiness. Alternatively or incombination, the reservoir may draw in air from a vent withoutwithdrawing any of the liquid.

The reservoir preserves the reactivity and composition of reagentstherein (e.g., the chemicals within the reservoir may exhibit little orno change in reactivity over 6 months or a year).

The flexible wall of the reservoir can limit or prevent leaching ofchemicals therethrough. The reservoir can be assembled independently ofa microfluidic cartridge and then secured to the microfluidic cartridge.

Exemplary Microfluidic Cartridge for Processing Polynucleotides

Referring to FIG. 10, a portion of an exemplary microfluidic cartridge200 suitable for use with the system described herein includes first,second, and third layers 205, 207, and 209 that define a microfluidicnetwork 201 having various components configured to process a samplethat includes one or more polynucleotides to be detected. Cartridge 200typically processes the sample by, among other aspects, increasing theconcentration of a polynucleotide to be determined and/or by reducingthe concentration of inhibitors relative to the concentration ofpolynucleotide to be determined. The various features of cartridge 200may be incorporated wholesale, or with suitable modification, intoalternative configurations of cartridge that carry out polynucleotideprocessing in conjunction with various other operations.

Fabrication of Cartridge

Microfluidic cartridge 200 can be fabricated as desired. Typically,layers 205, 207, and 209 can be formed of a polymeric material.Components of network 201 can typically be formed by molding (e.g., byinjection molding) layers 207, 209. Layer 205 can typically be aflexible polymeric material (e.g., a laminate) that can be secured(e.g., adhesively and/or thermally) to layer 207 to seal components ofnetwork 201. Layers 207 and 209 may be secured to one another usingadhesive. Other methods of cartridge fabrication suitable forapplication herein can be found described in U.S. provisional patentapplication Ser. No. 60/859,284, filed Nov. 14, 2006, and incorporatedherein by reference in its entirety.

Microfluidic Network

An exemplary arrangement of components of network 201 is as follows, asfurther described in U.S. Patent Application Publication No.2006/0166233, incorporated herein by reference.

Network 201 includes an inlet 202 by which sample material can beintroduced to the network and an output 236 by which a processed samplecan be removed (e.g., expelled by or extracted from) network 201. Achannel 204 extends between inlet 202 and a junction 255. A valve 206can be positioned along channel 204. A reservoir channel 240 extendsbetween junction 255 and an actuator 244. Gates 242 and 246 can bepositioned along channel 240. A channel 257 extends between junction 255and a junction 259. A valve 208 can be positioned along channel 257. Areservoir channel 246 extends between junction 259 and an actuator 248.Gates 250 and 252 can be positioned along channel 246. A channel 261extends between junction 259 and a junction 263. A valve 210 and ahydrophobic vent 212 can be positioned along channel 261. A channel 256extends between junction 263 and an actuator 254. A gate 258 can bepositioned along channel 256.

A channel 214 extends between junction 263 and a processing chamber 220,which has an inlet 265 and an outlet 267. A channel 228 extends betweenprocessing chamber outlet 267 and a waste reservoir 232. A valve 234 canbe positioned along channel 228. A channel 230 extends betweenprocessing chamber outlet 267 and output 236.

Particular components of microfluidic network 201 are further describedas follows.

Processing Chamber

Referring also to FIG. 11, processing chamber 220 includes a pluralityof particles (e.g., beads, microspheres) 218 configured to retainpolynucleotides of the sample under a first set of conditions (e.g., afirst temperature and/or first pH) and to release the polynucleotidesunder a second set of conditions (e.g., a second, higher temperatureand/or a second, more basic pH). Typically, the polynucleotides can beretained preferentially as compared to inhibitors that may be present inthe sample. Particles 218 can be configured as a retention member 216(e.g., a column) through which sample material (e.g., polynucleotides)must pass when moving between the inlet 265 and outlet 267 of processingregion 220.

A filter 219 prevents particles 218 from passing downstream ofprocessing region 220. A channel 287 connects filter 219 with outlet267. Filter 219 has a surface area within processing region 220 that canbe larger than the cross-sectional area of inlet 265. For example, insome embodiments, the ratio of the surface area of filter 219 withinprocessing chamber 220 to the cross-sectional area of inlet 265 (whichcross-sectional area is typically about the same as the cross-sectionalarea of channel 214) can be at least about 5 (e.g., at least about 10,at least about 20, at least about 30). In some embodiments, the surfacearea of filter 219 within processing region 220 can be at least about 1mm² (e.g., at least about 2 mm², at least about 3 mm²). In someembodiments, the cross-sectional area of inlet 265 and/or channel 214can be about 0.25 mm² or less (e.g., about 0.2 mm² or less, about 0.15mm² or less, about 0.1 mm² or less). The larger surface area presentedby filter 219 to material flowing through processing chamber 220 helpsprevent clogging of the processing region while avoiding significantincreases in the void volume (described hereinbelow) of the processingregion.

Particles 218 can be modified with at least one ligand that retainspolynucleotides (e.g., preferentially as compared to inhibitors).Typically, the ligands retain polynucleotides from liquids having a pHabout 9.5 or less (e.g., about 9.0 or less, about 8.75 or less, about8.5 or less). As a sample solution moves through processing chamber 220,polynucleotides can be retained while the liquid and other solutioncomponents (e.g., inhibitors) can be less retained (e.g., not retained)and exit the processing region. In general, the ligands releasepolynucleotides when the pH can be about 10 or greater (e.g., about 10.5or greater, about 11.0 or greater). Consequently, polynucleotides can bereleased from the ligand modified particles into the surrounding liquid.

Exemplary ligands on particles 218 include, for example, polyamides(e.g., poly-cationic polyamides such as poly-L-lysine, poly-D-lysine,poly-DL-ornithine) and PEI. Other ligands include, for example,intercalators, poly-intercalators, minor groove binders polyamines(e.g., spermidine), homopolymers and copolymers comprising a pluralityof amino acids, and combinations thereof. In some embodiments, theligands have an average molecular weight of at least about 5,000 Da(e.g., at least about 7,500 Da, of at least about 15,000 Da). In someembodiments, the ligands have an average molecular weight of about50,000 Da or less (e.g., about 35,000, or less, about 27,500 Da orless). In some embodiments, the ligand can be a poly-lysine ligandattached to the particle surface by an amide bond.

In certain embodiments, the ligands on the particles 218 can beresistant to enzymatic degradation, such as degradation by proteaseenzymes (e.g., mixtures of endo- and exo-proteases such as pronase) thatcleave peptide bonds. Exemplary protease resistant ligands include, forexample, poly-D-lysine and other ligands that can be enantiomers ofligands susceptible to enzymatic attack.

Particles 218 can typically be formed of a material to which the ligandscan be associated. Exemplary materials from which particles 218 can beformed include polymeric materials that can be modified to attach aligand. Typical polymeric materials provide or can be modified toprovide carboxylic groups and/or amino groups available to attachligands. Exemplary polymeric materials include, for example,polystyrene, latex polymers (e.g., polycarboxylate coated latex),polyacrylamide, polyethylene oxide, and derivatives thereof. Polymericmaterials that can used to form particles 218 are described in U.S. Pat.No. 6,235,313 to Mathiowitz et al., which patent is incorporated hereinby reference. Other materials include glass, silica, agarose, andamino-propyl-tri-ethoxy-silane (APES) modified materials.

Exemplary particles that can be modified with suitable ligands includecarboxylate particles (e.g., carboxylate modified magnetic beads(Sera-Mag Magnetic Carboxylate modified beads, Part #3008050250,Seradyn) and Polybead carboxylate modified microspheres available fromPolyscience, catalog no. 09850). In some embodiments, the ligandsinclude poly-D-lysine and the beads comprise a polymer (e.g.,polycarboxylate coated latex). In other embodiments, the ligands includePEI.

In general, the ratio of mass of particles to the mass ofpolynucleotides retained by the particles can be no more than about 25or more (e.g., no more than about 20, no more than about 10). Forexample, in some embodiments, about 1 gram of particles retains about100 milligrams of polynucleotides.

Typically, the total volume of processing chamber 220 (includingparticles 218) between inlet 265 and filter 219 can be about 15microliters or less (e.g., about 10 microliters or less, about 5microliters or less, about 2.5 microliters or less, about 2 microlitersor less). In an exemplary embodiment, the total volume of processingregion 220 can be about 2.3 microliters. In some embodiments, particles218 occupy at least about 10 percent (e.g., at least about 15 percent)of the total volume of processing region 220. In some embodiments,particles 218 occupy about 75 percent or less (e.g., about 50 percent orless, about 35 percent or less) of the total volume of processingchamber 220.

In some embodiments, the volume of processing chamber 220 that can befree to be occupied by liquid (e.g., the void volume of processingchamber 220 including interstices between particles 218) can be aboutequal to the total volume minus the volume occupied by the particles.Typically, the void volume of processing region 220 can be about 10microliters or less (e.g., about 7.5 microliters or less, about 5microliters or less, about 2.5 microliters or less, about 2 microlitersor less). In some embodiments, the void volume can be about 50nanoliters or more (e.g., about 100 nanoliters or more, about 250nanoliters or more). For example, in an exemplary embodiment, the totalvolume of processing chamber 220 can be about 2.3 microliters, thevolume occupied by particles can be about 0.3 microliters, and thevolume free to be occupied by liquid (void volume) can be about 2microliters.

Particles 218 typically have an average diameter of about 20 microns orless (e.g., about 15 microns or less, about 10 microns or less). In someembodiments, particles 218 have an average diameter of at least about 4microns (e.g., at least about 6 microns, at least about 8 microns).

In some embodiments, a volume of channel 287 between filter 219 andoutlet 267 can be substantially smaller than the void volume ofprocessing chamber 220. For example, in some embodiments, the volume ofchannel 287 between filter 219 and outlet 267 can be about 35% or less(e.g., about 25% or less, about 20% or less) of the void volume. In anexemplary embodiment, the volume of channel 287 between filter 219 andoutlet 267 can be about 500 nanoliters.

The particle density can typically be at least about 10⁸ particles permilliliter (e.g., about 10⁹ particles per milliliter). For example, aprocessing region with a total volume of about 1 microliter may includeabout 10³ beads.

Filter 219 typically has pores with a diameter smaller than the diameterof particles 218. In an exemplary embodiment, filter 219 has poreshaving an average width of about 8 microns, where particles 218 have anaverage diameter of about 10 microns.

In some embodiments, at least some (e.g., all) of the particles can bemagnetic. In alternative embodiments, few (e.g., none) of the particlesare magnetic.

In some embodiments, at least some (e.g., all) the particles can besolid. In some embodiments, at least some (e.g., all) the particles canbe porous (e.g., the particles may have channels extending at leastpartially within them).

Further components that may be found in microfluidic network 201 are asfollows.

Channels

Channels of microfluidic network 201 typically have at least onesub-millimeter cross-sectional dimension. For example, channels ofnetwork 201 may have a width and/or a depth of about 1 mm or less (e.g.,about 750 microns or less, about 500 microns, or less, about 250 micronsor less).

Valves

A valve can be a component that has a normally open state allowingmaterial to pass along a channel from a position on one side of thevalve (e.g., upstream of the valve) to a position on the other side ofthe valve (e.g., downstream of the valve). Upon actuation, the valvetransitions to a closed state that prevents material from passing alongthe channel from one side of the valve to the other. For example, inFIG. 10, valve 206 includes a mass 251 of a thermally responsivesubstance (TRS) that can be relatively immobile at a first temperatureand more mobile at a second temperature (e.g., a phase transitionmaterial (PTM) of a known melting point, typically about 60° C., orabout 75° C., or about 90° C., such as a paraffin wax, solder, etc.). Achamber 253 can be in gaseous communication with mass 251. Upon heatinggas (e.g., air) in chamber 253 and heating mass 251 of TRS to the secondtemperature, gas pressure within chamber 253 moves mass 251 into channel204 obstructing material from passing therealong. Other valves ofnetwork 201 have a similar structure and operate in a similar fashion asvalve 206.

A mass of TRS can be an essentially solid mass or an agglomeration ofsmaller particles that cooperate to obstruct the passage. Examples ofTRS's include a eutectic alloy (e.g., a solder), wax (e.g., an olefin),polymers, plastics, and combinations thereof. The first and secondtemperatures can be insufficiently high to damage materials, such aspolymer layers of cartridge 200. Generally, the second temperature canbe less than about 90° C. and the first temperature can be less than thesecond temperature (e.g., about 70° C. or less).

A gate can be a component that can have a closed state that does notallow material to pass along a channel from a position on one side ofthe gate to another side of the gate, and an open state that does allowmaterial to pass along a channel from a position on one side of the gateto another side of the gate. Actuation of an open gate can transitionthe gate to a closed state in which material is not permitted to passfrom one side of the gate (e.g., upstream of the gate) to the other sideof the gate (e.g., downstream of the gate). Upon actuation, a closedgate can transition to an open state in which material is permitted topass from one side of the gate (e.g., upstream of the gate) to the otherside of the gate (e.g., downstream of the gate). For example, gate 242in FIG. 10 includes a mass 271 of TRS positioned to obstruct passage ofmaterial between junction 255 and channel 240. Upon heating mass 271 toa second temperature, the mass changes state (e.g., by melting, bydispersing, by fragmenting, and/or dissolving) to permit passage ofmaterial between junction 255 and channel 240.

In various embodiments, a microfluidic network 201 can include a narrowgate 380 as shown in FIG. 12A where a gate loading channel 382 used forloading wax from a wax loading hole 384 to a gate junction 386 can benarrower (e.g., approximately 150 μm wide and 100 microns deep). Anupstream channel 388 as well as a downstream channel 390 of the gatejunction 386 can be made wide (e.g., ˜500 μm) and deep (e.g., ˜500 μm)to help ensure the wax stops at the gate junction 386. The amount ofgate material melted and moved out of the gate junction 386 may beminimized for optimal gate 380 opening. As an off-cartridge heater maybe used to melt the thermally responsive substance in gate 380, amisalignment of the heater could cause the wax in the gate loadingchannel 382 to be melted as well. Therefore, narrowing the dimension ofthe loading channel may increase reliability of gate opening. In thecase of excessive amounts of wax melted at the gate junction 386 andgate loading channel 382, the increased cross-sectional area of thedownstream channel 390 adjacent to the gate junction 386 can prevent waxfrom clogging the downstream channel 390 during gate 380 opening. Thedimensions of the upstream channel 388 at the gate junction 386 can bemade similar to the downstream channel 390 to ensure correct wax loadingduring gate fabrication.

In various embodiments, the gate can be configured to minimize theeffective area or footprint of the gate within the network, such as bentgate 392 as shown in FIG. 12B. Minimizing the effective area orfootprint of the gate within the network can increase the density of agiven microfluidic network and can thereby reduce the cost per part,provide for a more compact network, minimize network channel length orvolume, or the like. Still other configurations are possible, though notexplicitly shown in the drawings, according to specific layouts ofmicrofluidic network.

In the microfluidic cartridge of FIG. 10, the portion of channel 240between gates 242 and 246 forms a fluid reservoir 279 configured to holda liquid (e.g., water, an organic liquid, or combination thereof).During storage, gates 242 and 246 limit (e.g., prevent) evaporation ofliquid within the fluid reservoir. During operation of cartridge 200,the liquid of reservoir 279 can typically be used as a wash liquid toremove inhibitors from processing region 220 while leavingpolynucleotides associated with particles 218 (FIG. 11). Typically, thewash liquid can be a solution having one or more additional components(e.g., a buffer, chelator, surfactant, a detergent, a base, an acid, ora combination thereof). Exemplary solutions include, for example, asolution of 10-50 mM Tris at pH 8.0, 0.5-2 mM EDTA, and 0.5%-2% SDS, asolution of 10-50 mM Tris at pH 8.0, 0.5 to 2 mM EDTA, and 0.5%-2%Triton X-100.

The portion of channel 247 between gates 250 and 252 form a fluidreservoir 281 configured like reservoir 279 to hold a liquid (e.g., asolution) with limited or no evaporation. During operation of cartridge200, the liquid of reservoir 281 can typically be used as a releaseliquid into which polynucleotides that had been retained by particles218 can be released. An exemplary release liquid can be a hydroxidesolution (e.g., a NaOH solution) having a concentration of, for example,between about 2 mM hydroxide (e.g., about 2 mM NaOH) and about 500 mMhydroxide (e.g., about 500 mM NaOH). In some embodiments, liquid inreservoir 281 can be a hydroxide solution having a concentration ofabout 25 mM or less (e.g., a hydroxide concentration of about 15 mM).

Reservoirs 279, 281 typically each independently hold at least about0.375 microliters of liquid (e.g., at least about 0.750 microliters, atleast about 1.25 microliters, at least about 2.5 microliters). In someembodiments, reservoirs 279, 281 each independently hold about 7.5microliters or less of liquid (e.g., about 5 microliters or less, about4 microliters or less, about 3 microliters or less).

Actuators

An actuator can be a component that provides a gas pressure that canmove material (e.g., sample material and/or reagent material) betweenone location in a network e.g., network 201, and another location. Forexample, referring to FIG. 13, actuator 244 includes a chamber 272having a mass 273 of thermally expansive material (TEM) therein. Whenheated, the TEM expands decreasing the free volume within chamber 272and pressurizing the gas (e.g., air) surrounding mass 273 within chamber272. Typically, gates such as gates 246 and 242 in network 201 can beactuated with actuator 244. Consequently, the pressurized gas drivesliquid in fluid reservoir 279 towards junction 255. In some embodiments,actuator 244 can generate a pressure differential of more than about 3psi (e.g., at least about 4 psi, at least about 5 psi) between theactuator and junction 255.

In one embodiment, shown in FIG. 13, the TEM includes a plurality ofsealed liquid reservoirs (e.g., spheres) 275 dispersed within a carrier277. Typically, the liquid can be a high vapor pressure liquid (e.g.,isobutane and/or isopentane) sealed within a casing (e.g., a polymericcasing formed of monomers such as vinylidene chloride, acrylonitrile andmethylmethacrylate). Carrier 277 has properties (e.g., flexibilityand/or an ability to soften (e.g., melt) at higher temperatures) thatpermit expansion of the reservoirs 275 without allowing the reservoirsto pass along channel 240. In some embodiments, carrier 277 can be a wax(e.g., an olefin) or a polymer with a suitable glass transitiontemperature. Typically, the reservoirs make up at least about 25 weightpercent (e.g., at least about 35 weight percent, at least about 50weight percent) of the TEM. In some embodiments, the reservoirs make upabout 75 weight percent or less (e.g., about 65 weight percent or less,about 50 weight percent or less) of the TEM. Suitable sealed liquidreservoirs can be obtained from Expancel (available from Akzo Nobel).

When the TEM can be heated (e.g., to a temperature of at least about 50°C. (e.g., to at least about 75° C., or at least about 90° C.)), theliquid vaporizes and increases the volume of each sealed reservoir andof mass 273. Carrier 277 softens allowing mass 273 to expand. Typically,the TEM can be heated to a temperature of less than about 150° C. (e.g.,about 125° C. or less, about 110° C. or less, about 100° C. or less)during actuation. In some embodiments, the volume of the TEM expands byat least about 5 times (e.g., at least about 10 times, at least about 20times, at least about 30 times).

Vents

A hydrophobic vent (e.g., vent 212) can be a structure that permits gasto exit a channel while limiting (e.g., preventing) liquid from exitingthe channel. Typically, hydrophobic vents include a layer of poroushydrophobic material (e.g., a porous filter such as a porous hydrophobicmembrane from Osmonics) that defines a wall of the channel. As describedhereinbelow, hydrophobic vents can be used to position a microdroplet ofsample at a desired location within network 201.

The hydrophobic vents of the present technology are preferablyconstructed so that the amount of air that escapes through them can bemaximized while minimizing the volume of the channel below the ventsurface. Accordingly, it is preferable that the vent can be constructedso as to have a hydrophobic membrane of large surface area and a shallowcross section of the microchannel below the vent surface.

Hydrophobic vents typically have a length of at least about 2.5 mm(e.g., at least about 5 mm, at least about 7.5 mm) along a channel. Thelength of the hydrophobic vent can typically be at least about 5 times(e.g., at least about 10 times, at least about 20 times) larger than adepth of the channel within the hydrophobic vent. For example, in someembodiments, the channel depth within the hydrophobic vent can be about300 microns or less (e.g., about 250 microns or less, about 200 micronsor less, about 150 microns or less).

The depth of the channel within the hydrophobic vent can typically beabout 75% or less (e.g., about 65% or less, about 60% or less) of thedepth of the channel upstream and downstream of the hydrophobic vent.For example, in some embodiments the channel depth within thehydrophobic vent can be about 150 microns and the channel depth upstreamand downstream of the hydrophobic vent can be about 250 microns.

A width of the channel within the hydrophobic vent can typically be atleast about 25% wider (e.g., at least about 50% wider) than a width ofthe channel upstream from the vent and downstream from the vent. Forexample, in an exemplary embodiment, the width of the channel within thehydrophobic vent can be about 400 microns and the width of the channelupstream and downstream from the vent can be about 250 microns.

In use, cartridge 200 can typically be thermally associated with anarray of heat sources configured to operate various components (e.g.,valves, gates, actuators, and processing region 220) of the cartridge.In some embodiments, the heat sources can be controlled by a processorin a system such as that further described herein, which operates toreceive and to monitor the cartridge during use. The processor (e.g., amicroprocessor) is configured to actuate the heat sources individuallyand at different times, according to a desired protocol. Processorsconfigured to operate microfluidic cartridges, suitable for use or formodification for use herein, are described in U.S. application Ser. No.09/819,105, filed Mar. 28, 2001 (now U.S. Pat. No. 7,010,391), whichpatent is incorporated herein by reference. In other embodiments, theheat sources can be integral with the cartridge itself.

Cartridge 200 may be operated as follows. Valves of network 201 can befabricated in an open state. Gates of network 201 can be fabricated in aclosed state. A fluid sample, such as a biological sample as furtherdescribed herein, comprising polynucleotides can be introduced tonetwork 201 via inlet 202. For example, sample can be introduced with asyringe having a Luer fitting. The syringe provides pressure toinitially move the sample within network 201. Sample passes alongchannels 204, 257, 261, and 214 to inlet 265 of processing region 220.The sample passes through processing region 220, exits via outlet 267,and passes along channel 228 to waste chamber 232. When the trailingedge (e.g., the upstream liquid-gas interface) of the sample reacheshydrophobic vent 212, pressure provided by the introduction device(e.g., the syringe) can be released from network 201 stopping furthermotion of the sample.

Typically, the amount of sample introduced can be about 500 microlitersor less (e.g., about 250 microliters or less, about 100 microliters orless, about 50 microliters or less, about 25 microliters or less, about10 microliters or less). In some embodiments, the amount of sample canbe about 2 microliters or less (e.g., about 0.5 microliters or less).

Polynucleotides entering processing region 220 pass through intersticesbetween the particles 218. Polynucleotides of the sample contactretention member 216 and can be preferentially retained as compared toliquid of the sample, and certain other sample components (e.g.,inhibitors). Typically, retention member 220 retains at least about 50%of polynucleotides (e.g., at least about 75%, at least about 85%, atleast about 90%) of the polynucleotides present in the sample thatentered processing region 220. Liquid of the sample and inhibitorspresent in the sample exit the processing region 220 via outlet 267 andenter waste chamber 232. Processing region 220 can typically be at atemperature of about 50° C. or less (e.g., 30° C. or less) duringintroduction of the sample.

Processing continues by washing retention member 216 with liquid ofreservoir 279 to separate remaining inhibitors from polynucleotidesretained by retention member 216. To wash retention member 216, valve206 can be closed and gates 242, 246 of first reservoir 240 can beopened. Actuator 244 can be actuated to move wash liquid withinreservoir 279 along channels 257, 261, and 214, through processingregion 220, and into waste reservoir 232. The wash liquid moves samplethat may have remained within channels 204, 257, 261, and 214 throughthe processing region and into waste chamber 232. Once the trailing edgeof the wash liquid reaches vent 212, the gas pressure generated byactuator 244 can be vented and further motion of the liquid can bestopped.

The volume of wash liquid moved by actuator 244 through processingregion 220 can typically be at least about 2 times the void volume ofprocessing region 220 (e.g., at least about 3 times the void volume) andcan be about 10 times the void volume or less (e.g., about 5 times thevoid volume or less). Processing region can typically be at atemperature of about 50° C. or less (e.g., 30° C. or less) duringwashing. Exemplary wash fluids include liquids described with respect toreservoirs 279 and 281, herein.

Processing continues by releasing polynucleotides from retention member216. Typically, wash liquid from reservoir 279 can be replaced withrelease liquid (e.g., an hydroxide solution) from reservoir 281 beforereleasing the polynucleotides. Valve 208 can be closed and gates 250,252 can be opened. Actuator 248 can be actuated, thereby moving releaseliquid within reservoir 281 along channels 261, 214 and into processingregion 220 and in contact with retention member 216. When the trailingedge of release liquid from reservoir 281 reaches hydrophobic vent 212,pressure generated by actuator 248 can be vented stopping the furthermotion of the liquid. The volume of liquid moved by actuator 248 throughprocessing region 220 can typically be at least about equal to the voidvolume of the processing region 220 (e.g., at least about 2 times thevoid volume) and can be about 10 times the void volume or less (e.g.,about 5 times the void volume or less).

Once retention member 216 with retained polynucleotides has beencontacted with liquid from reservoir 281, a releasing step can typicallybe performed. Typically, the releasing includes heating release liquidpresent within processing region 216. Generally, the liquid can beheated to a temperature insufficient to boil liquid in the presence ofthe retention member. In some embodiments, the temperature can be 100°C. or less (e.g., less than 100° C., about 97° C. or less). In someembodiments, the temperature can be about 65° C. or more (e.g., about75° C. or more, about 80° C. or more, about 90° C. or more). In someembodiments, the temperature is maintained for about 1 minute or more(e.g., about 2 minutes or more, about 5 minutes or more, about 10minutes or more). In some embodiments, the temperature can be maintainedfor about 30 minutes (e.g., about 15 minutes or less, about 10 minutesor less, about 5 minutes or less). In an exemplary embodiment,processing region 220 can be heated to between about 65 and 90° C.(e.g., to about 70° C.) for between about 1 and 7 minutes (e.g., forabout 2 minutes). Such temperatures and times vary according to thesample and can be chosen accordingly by one of ordinary skill in theart.

The polynucleotides can be released into the liquid present in theprocessing region 220 (e.g., the polynucleotides can typically bereleased into an amount of release liquid having a volume about the sameas the void volume of the processing region 220). Typically, thepolynucleotides can be released into about 10 microliters or less (e.g.,about 5 microliters or less, about 2.5 microliters or less) of liquid.

In certain embodiments, the ratio of the volume of original sample movedthrough the processing region 220 to the volume of liquid into which thepolynucleotides can be released can be at least about 10 (e.g., at leastabout 50, at least about 100, at least about 250, at least about 500, atleast about 1000). In some embodiments, polynucleotides from a samplehaving a volume of about 2 ml can be retained within the processingregion, and released into about 4 microliters or less (e.g., about 3microliters or less, about 2 microliters or less, about 1 microliter orless) of liquid.

The liquid into which the polynucleotides can be released typicallyincludes at least about 50% (e.g., at least about 75%, at least about85%, at least about 90%) of the polynucleotides present in the samplethat entered processing region 220. The concentration of polynucleotidespresent in the release liquid may be higher than in the original samplebecause the volume of release liquid can typically be less than thevolume of the original liquid sample moved through the processingregion. For example the concentration of polynucleotides in the releaseliquid may be at least about 10 times greater (e.g., at least about 25times greater, at least about 100 times greater) than the concentrationof polynucleotides in the sample introduced to cartridge 200. Theconcentration of inhibitors present in the liquid into which thepolynucleotides can be released can generally be less than concentrationof inhibitors in the original fluid sample by an amount sufficient toincrease the amplification efficiency for the polynucleotides.

The time interval between introducing the polynucleotide containingsample to processing region 220 and releasing the polynucleotides intothe release liquid can typically be about 15 minutes or less (e.g.,about 10 minutes or less, about 5 minutes or less).

Liquid including the released polynucleotides may be removed from theprocessing region 220 as follows. Valves 210 and 234 can be closed.Gates 238 and 258 can be opened. Actuator 254 can be actuated togenerate pressure that moves liquid and polynucleotides from processingregion 220, into channel 230, and toward outlet 236. The liquid withpolynucleotides can be removed using, for example, a syringe orautomated sampling device. Depending upon the liquid in contact withretention member 216 during polynucleotide release, the solution withreleased polynucleotide may be neutralized with an amount of buffer(e.g., an equal volume of 25-50 mM Tris-HCl buffer pH 8.0).

While releasing the polynucleotides has been described as including aheating step, the polynucleotides may be released without heating. Forexample, in some embodiments, the liquid of reservoir 281 has an ionicstrength, pH, surfactant concentration, composition, or combinationthereof that releases the polynucleotides from the retention member atambient temperature, without need for additional heating.

While the polynucleotides have been described as being released into asingle volume of liquid present within processing region 220, otherconfigurations can be used. For example, polynucleotides may be releasedwith the concomitant (stepwise or continuous) introduction of fluid intoand/or through processing region 220. In such embodiments, thepolynucleotides may be released into liquid having a volume of about 10times or less (e.g., about 7.5 times or less, about 5 times or less,about 2.5 times or less, about 2 times or less) than the void volume ofthe processing region 220.

While reservoirs 279, 281 have been described as holding liquids betweenfirst and second gates, other configurations can be used. For example,liquid for each reservoir may be held within a pouch (e.g., a blisterpack, as further described herein) isolated from network 201 by agenerally impermeable membrane. The pouch can be configured so that auser can rupture the membrane driving liquid into reservoirs 279, 281where actuators 244, 248 can move the liquid during use.

While processing regions have been described as having microliter scaledimensions, other dimensions can be used. For example, processingregions with surfaces (e.g., particles) configured to preferentiallyretain polynucleotides as opposed to inhibitors may have large volumes(e.g., many tens of microliters or more, at least about 1 milliliter ormore). In some embodiments, the processing region has a bench-top scale.

While processing region 220 has been described as having a retentionmember formed of multiple surface-modified particles, otherconfigurations can be used. For example, in some embodiments, processingregion 220 includes a retention member configured as a porous member(e.g., a filter, a porous membrane, or a gel matrix) having multipleopenings (e.g., pores and/or channels) through which polynucleotidespass. Surfaces of the porous member can be modified to preferentiallyretain polynucleotides. Filter membranes available from, for example,Osmonics, can be formed of polymers that may be surface-modified andused to retain polynucleotides within processing region 220. In someembodiments, processing region 220 includes a retention memberconfigured as a plurality of surfaces (e.g., walls or baffles) throughwhich a sample passes. The walls or baffles can be modified topreferentially retain polynucleotides.

While processing region 220 has been described as a component of amicrofluidic network, other configurations can be used. For example, insome embodiments, the retention member can be removed from a processingregion for processing elsewhere. For example, the retention member maybe contacted with a mixture comprising polynucleotides and inhibitors inone location and then moved to another location at which thepolynucleotides can be removed from the retention member.

While reservoirs 275 have been shown as dispersed within a carrier,other configurations may be used. For example, reservoirs 275 can beencased within a flexible enclosure (e.g., a membrane, for example, anenclosure such as a sack). In some embodiments, reservoirs can be loosewithin chamber 272. In such embodiments, actuator 244 may include aporous member having pores too small to permit passage of reservoirs 275but large enough to permit gas to exit chamber 272.

Exemplary Microfluidic Cartridge Having a Lysing Chamber

Further microfluidic cartridge with various components are described inU.S. provisional application No. 60/553,553 filed Mar. 17, 2004 byParunak, et al., and patent application publication no. 2005-0084424,which applications are incorporated herein by reference.

While microfluidic cartridges have been described that are configured toreceive polynucleotides already released from cells, microfluidiccartridges for use herein can also be configured to releasepolynucleotides from cells (e.g., by lysing the cells). For example,referring to FIGS. 14A, 14B, 15A, and 15B, a microfluidic cartridge 300includes a sample lysing chamber 302 in which cells can be lysed torelease polynucleotides therein. Microfluidic cartridge 300 furtherincludes substrate layers L1-L3, a microfluidic network 304 (onlyportions of which are seen in FIG. 14A), and liquid reagent reservoirsR1-R4. Liquid reagent reservoirs R1-R4 hold liquid reagents (e.g., forprocessing sample material) and can be connected to network 304 byreagent ports RP1-RP4. Microfluidic cartridge 300 can therefore be aself-contained environment that comprises all reagents and materialsnecessary to perform steps of work-up, cell-lysis, polynucleotideisolation, pre-amplification processing, amplification, and detection ofa sample. In some embodiments, a sample is introduced having one or moreof the required reagents mixed therewith; in which case the remainingreagents are stored on the cartridge. Reagents and other materials maybe stored on cartridge 300 in liquid reagent reservoirs R1-R4,microfluidic channels or chambers in a microfluidic network, and/or in alysing chamber 302.

Network 304 can be substantially defined between layers L2 and L3 butextends in part between all three layers L1-L3. Microfluidic network 304includes various microfluidic components as further described herein,including channels Ci, valves Vi, double valves V′i, gates Gi, mixinggates MGi, vents Hi, gas actuators (e.g., pumps) Pi, a first processingregion B1, a second processing region B2, detection zones Di, air ventsAVi, and waste zones Wi.

FIGS. 15A, 15B, show two complementary halves of an exemplarymicrofluidic network 304. It would be understood by one of ordinaryskill in the art, that the division of the network into two separatehalves is arbitrary, and purely for ease of illustration. Thearrangement of components shown in FIGS. 15A, 15B is exemplary; it wouldbe understood by one of ordinary skill in the art that other sucharrangements, such as different geometric arrangements of the samecomponents, or different arrangements of different components may beconstructed by one of ordinary skill in the art, to accomplish the stepsdescribed herein.

Components of network 304 can typically be thermally actuated. As seenin FIG. 16, an exemplary heat source network 312 includes heat sources(e.g., resistive heat sources) having locations that correspond tovarious thermally actuated components of microfluidic network 304. Forexample, the locations of heat sources HPi correspond to the locationsof actuators Pi, the locations of heat sources HGi correspond tolocations of gates Gi and mixing gates MGi, the locations of heatsources HVi correspond to the locations of valves Vi and double valvesV′i, and the locations of heat sources HDi correspond to the locationsof processing chambers Di, all of network 304. In use, the components ofcartridge 300 can be disposed in thermal contact with corresponding heatsources of network 312, which can typically be operated using aprocessor as described above for cartridge 200. Heat source network 312can be integral with or separate from cartridge 300 as described forcartridge 200. For example, heat source network 312 can be integratedinto a heater module 2020, such as beneath the receiving bay 2014 in amanner that it aligns with a network of a cartridge disposed therein.

Further components of exemplary microfluidic cartridge 300 are asfollows.

Air Vents

Air vents AVi can allow gas (e.g., air) displaced by the movement ofliquids within network 304 to be vented so that pressure buildup doesnot inhibit desired movement of the liquids. For example, air vent AV2permits liquid to move along channel C14 and into channel C16 by ventinggas downstream of the liquid through vent AV2.

Valves

Valves Vi can have a normally open state allowing material to pass alonga channel from a position on one side of the valve (e.g., upstream ofthe valve) to a position on the other side of the valve (e.g.,downstream of the valve). The valves Vi can have a similar structure tovalves of microfluidic cartridge 200, as further described herein.

As seen in FIGS. 17 and 18, double valves V′i can also have a normallyopen state allowing material to pass along a channel from a position onone side of the valve (e.g., upstream of the valve) to a position on theother side of the valve (e.g., downstream of the valve). Taking doublevalve V11′ of FIGS. 17 and 18 as an example, double valves Vi′ includefirst and second masses 314, 316 of a TRS (e.g., a eutectic alloy orwax) spaced apart from one another on either side of a channel (e.g.,channel C14). Typically, the TRS masses 314, 316 can be offset from oneanother (e.g., by a distance of about 50% of a width of the TRS massesor less). Material moving through the open valve passes between thefirst and second TRS masses 314, 316. Each TRS mass 314, 316 can beassociated with a respective chamber 318, 320, which typically includesa gas (e.g., air).

The TRS masses 314, 316 and chambers 318, 320 of double valve Vi′ can bein thermal contact with a corresponding heat source HV11′ of heat sourcenetwork 312. Actuating heat source HV11′ causes TRS masses 314, 316 totransition to a more mobile second state (e.g., a partially meltedstate) and increases the pressure of gas within chambers 318, 320. Thegas pressure drives TRS masses 314, 316 across channel C11 and closesvalve HV11′ (FIG. 18). Typically, masses 314, 316 at least partiallycombine to form a mass 322 that obstructs channel C11.

Returning to FIGS. 15A, 15B, gates Gi can have a normally closed statethat does not allow material to pass along a channel from a position onone side of the gate to another side of the gate. Gates Gi can have asimilar structure as described for gates of cartridge 200.

As seen in FIGS. 19A-19D for an exemplary portion of a microfluidicnetwork, mixing gates MGi can allow two volumes of liquid to be combined(e.g., mixed) within network 304. Mixing gates MGi are described furtherbelow.

Actuators

Actuators Pi can provide a gas pressure to move material (e.g., samplematerial and/or reagent material) between one location of network 304and another location. Actuators Pi can be similar in form to actuatorsof cartridge 200. For example, each actuator Pi includes a chamber witha mass 273 of TEM that can be heated to pressurize gas within thechamber. Each actuator Pi includes a corresponding gate Gi (e.g., gateG2 of actuator P1) that prevents liquid from entering the chamber of theactuator. The gate can typically be actuated (e.g., opened) to allowpressure created in the chamber of the actuator to enter themicrofluidic network.

Waste Chambers

Waste chambers Wi can receive waste (e.g., overflow) liquid resultingfrom the manipulation (e.g., movement and/or mixing) of liquids withinnetwork 304. Typically, each waste chamber Wi has an associated air ventthat allows gas displaced by liquid entering the chamber to be vented.

Processing Regions

First processing region B1 of network 304 can be a component that allowspolynucleotides to be concentrated and/or separated from inhibitors of asample. Processing region B1 can be configured and operated asprocessing region 220 of cartridge 200. In some embodiments, firstprocessing region B1 includes a retention member (e.g., multipleparticles (e.g., microspheres or beads), a porous member, multiplewalls) having at least one surface modified with one or more ligands asdescribed for processing region 220. For example, the ligand can includeone or more polyamides (e.g., poly-cationic polyamides such aspoly-L-lysine, poly-D-lysine, poly-DL-ornithine), or polyethyleneimine.In some embodiments, particles of the retention member can be disposedin lysing chamber 302 and can be moved into processing region B1 alongwith sample material.

Second processing region B2 can be a component that allows material(e.g., sample material) to be combined with compounds (e.g., reagents)for determining the presence of one or more polynucleotides. In someembodiments, the compounds include one or more PCR reagents (e.g.,primers, control plasmids, and polymerase enzymes).

Lyophilized Particles

In some embodiments, the compounds for determining the presence of oneor more polynucleotides can be stored within a processing region such asB2 as one or more lyophilized particles (e.g., pellets). The particlesgenerally have a room temperature (e.g., about 20° C.) shelf-life of atleast about 6 months (e.g., at least about 12 months). Liquid enteringthe second processing region B2 dissolves (e.g., reconstitutes) thelyophilized compounds.

Typically, the lyophilized particle(s) of processing region B2 have anaverage volume of about 5 microliters or less (e.g., about 4 microlitersor less, about 3 microliters or less, about 2 microliters or less). Insome embodiments, the lyophilized particle(s) of processing region B2have an average diameter of about 4 mm or less (e.g., about 3 mm orless, about 2 mm or less) In an exemplary embodiment the lyophilizedparticle(s) have an average volume of about 2 microliters and an averagediameter of about 1.35 mm. In other embodiments, the lyophilizedparticles may have a diameter of about 5 mm or less (e.g., about 2.5 mmor less, about 1.75 mm or less).

Lyophilized particles for determining the presence of one or morepolynucleotides typically include multiple compounds. In someembodiments, the lyophilized particles include one or more compoundsused in a reaction for determining the presence of a polynucleotideand/or for increasing the concentration of the polynucleotide. Forexample, lyophilized particles can include one or more enzymes foramplifying a polynucleotide, as by PCR.

Exemplary lyophilized particles include exemplary reagents for theamplification of polynucleotides associated with group B streptococcus(GBS) bacteria. In some embodiments, the lyophilized particles includeone or more of a cryoprotectant, one or more salts, one or more primers(e.g., GBS Primer F and/or GBS Primer R), one or more probes (e.g., GBSProbe—FAM), one or more internal control plasmids, one or morespecificity controls (e.g., Streptococcus pneumoniae DNA as a controlfor PCR of GBS), one or more PCR reagents (e.g., dNTPs and/or dUTPs),one or more blocking or bulking agents (e.g., non-specific proteins(e.g., bovine serum albumin (BSA), RNAseA, or gelatin), and a polymerase(e.g., glycerol-free Taq Polymerase). Of course, other components (e.g.,other primers and/or specificity controls) can be used for amplificationof other polynucleotides.

Cryoprotectants generally help increase the stability of the lyophilizedparticles and help prevent damage to other compounds of the particles(e.g., by preventing denaturation of enzymes during preparation and/orstorage of the particles). In some embodiments, the cryoprotectantincludes one or more sugars (e.g., one or more dissacharides (e.g.,trehalose, melezitose, raffinose)) and/or one or more poly-alcohols(e.g., mannitol, sorbitol).

Lyophilized particles can be prepared as desired. A method for makinglyophilized particles includes forming a solution of reagents of theparticle and a cryoprotectant (e.g., a sugar or poly-alcohol).Typically, compounds of the lyophilized particles can be combined with asolvent (e.g., water) to make a solution, which can be then placed(e.g., dropwise, in discrete aliquots (e.g., drops) such as by pipette)onto a chilled hydrophobic surface (e.g., a diamond film or apolytetrafluorethylene surface). In general, the temperature of thesurface can be reduced to near the temperature of liquid nitrogen (e.g.,about −150° F. or less, about −200° F. or less, about −275° F. or less),such as by use of a cooling bath of a cryogenic agent directlyunderneath. The solution can be dispensed without contacting thecryogenic agent. The solution freezes as discrete particles. The frozenparticles can be subjected to a vacuum, typically while still frozen,for a pressure and time sufficient to remove the solvent (e.g., bysublimation) from the pellets. Such methods are further described ininternational patent application publication no. WO 2006/119280,incorporated herein by reference.

In general, the concentrations of the compounds in the solution fromwhich the particles are made can be higher than when reconstituted inthe microfluidic cartridge. Typically, the ratio of the solutionconcentration to the reconstituted concentration can be at least about 3(e.g., at least about 4.5). In some embodiments, the ratio can be about6.

An exemplary solution for preparing lyophilized pellets for use in theamplification of polynucleotides indicative of the presence of GBS canbe made by combining a cryoprotecant (e.g., 120 mg of trehalose as drypowder), a buffer solution (e.g., 48 microliters of a solution of 1MTris at pH 8.4, 2.5M KCl, and 200 mM MgCl₂), a first primer (e.g., 1.92microliters of 500 micromolar GBS Primer F (Invitrogen)), a secondprimer (e.g., 1.92 microliters of 500 micromolar GBS Primer R(Invitrogen)), a probe (e.g., 1.92 microliters of 250 micromolar GBSProbe—FAM (IDT/Biosearch Technologies)), a control probe (e.g., 1.92microliters of 250 micromolar Cal Orange 560 (Biosearch Technologies)),a template plasmid (e.g., 0.6 microliters of a solution of 10⁵ copiesplasmid per microliter), a specificity control (e.g., 1.2 microliters ofa solution of 10 nanograms per microliter (e.g., about 5,000,000 copiesper microliter) Streptococcus pneumoniae DNA (ATCC)), PCR reagents(e.g., 4.8 microliters of a 100 millimolar solution of dNTPs (Epicenter)and 4. microliters of a 20 millimolar solution of dUTPs (Epicenter)), abulking agent (e.g., 24 microliters of a 50 milligram per millilitersolution of BSA (Invitrogen)), a polymerase (e.g., 60 microliters of a 5U per microliter solution of glycerol-free Taq Polymerase(Invitrogen/Eppendorf)) and a solvent (e.g., water) to make about 400microliters of solution. About 200 aliquots of about 2 microliters eachof this solution can be frozen and desolvated as described above to make200 pellets. When reconstituted, the 200 particles make a PCR reagentsolution having a total volume of about 2.4 milliliters.

Reagent Reservoirs

As seen in FIG. 14, reagent reservoirs Ri can be configured to holdliquid reagents (e.g., water, buffer solution, hydroxide solution)separated from network 304 until ready for use. Reservoirs R1 include anenclosure 329 that defines a sealed space 330 for holding liquids. Eachspace 330 can be separated from reagent port RPi and network 304 by alower wall 333 of enclosure 329. A capping material 341 (e.g., alaminate, adhesive, or polymer layer) may overlie an upper wall of theenclosure.

A portion of enclosure 329 can be formed as an actuation mechanism(e.g., a piercing member 331) oriented toward the lower wall 333 of eachenclosure. When cartridge 300 can be used, reagent reservoirs Ri can beactuated by depressing piercing member 331 to puncture wall 333.Piercing member 331 can be depressed by a user (e.g., with a thumb) orby the operating system used to operate cartridge 300.

Wall 333 can typically be formed of a material having a low vaportransmission rate (e.g., Aclar, a metallized (e.g. aluminum) laminate, aplastic, or a foil laminate) that can be ruptured or pierced. Reservoir330 holds an amount of liquid suited for cartridge 300. For example, thereservoir may hold up to about 200 microliters. The piercing member 331may account for a portion (e.g., up to about 25%) of that volume. Thematerial of the laminate inside the blister that may touch corrosivereagent such as basic sodium hydroxide should not corrode even after sixto twelve months of exposure.

In general, reservoirs Ri can be formed and filled as desired. Forexample, the upper wall of the enclosure can be sealed to the lower wall333 (e.g., by adhesive and/or thermal sealing). Liquid can be introducedinto the reservoir by, for example, an opening at the lower end of thepiercing member 331. After filling, the opening can be sealed (e.g., byheat sealing through the localized application of heat or by theapplication of a sealing material (e.g., capping material 341)).

When wall 333 can be punctured, fluid from the reservoir enters network333. For example, as seen in FIGS. 14 and 15, liquid from reservoir R2enters network 304 by port RP2 and travels along a channel C2. Gate G3prevents the liquid from passing along channel C8. Excess liquid passesalong channel C7 and into waste chamber W2. When the trailing edge ofliquid from reservoir R2 passes hydrophobic vent H2, pressure createdwithin the reservoir can be vented stopping further motion of theliquid. Consequently, network 304 receives an aliquot of liquid reagenthaving a volume defined by the volume of channel C2 between a junctionJ1 and a junction J2. When actuator P1 can be actuated, this aliquot ofreagent can be moved further within network 304. Reagent reservoirs R1,R3, and R4 can be associated with corresponding channels, hydrophobicvents, and actuators.

In the configuration shown, reagent reservoir R1 typically holds arelease liquid (e.g., a hydroxide solution as described above forcartridge 200) for releasing polynucleotides retained within processingregion B1. Reagent reservoir R2 typically holds a wash liquid (e.g., abuffer solution as described above for cartridge 200) for removingun-retained compounds (e.g., inhibitors) from processing region B1 priorto releasing the polynucleotides. Reagent reservoir R3 typically holds aneutralization buffer (e.g., 25-50 mM Tris-HCl buffer at pH 8.0).Reagent reservoir R4 typically holds deionized water.

While reservoirs have been shown as having a piercing member formed of awall of the reservoir, other configurations are possible. For example,in some embodiments, the reservoir includes a needle-like piercingmember that extends through an upper wall of the reservoir into thesealed space toward a lower wall of the reservoir. The upper wall of thereservoir may be sealed at the needle-like piercing member (e.g., withan adhesive, an epoxy). In use, the upper wall can be depressed drivingthe piercing member through the lower wall forcing liquid in the sealedspace to enter a microfluidic network.

While reservoirs have been described as including an actuation mechanism(e.g., a piercing member), other configurations are possible. Forexample, in some embodiments, a lower wall of the sealed space of thereservoir includes a weakened portion that overlies an opening to amicrofluidic network. The lower wall material (e.g., laminate, polymerfilm, or foil) that overlies the opening can be thick enough to preventloss of the liquid within the sealed space but thin enough to ruptureupon the application of pressure to the liquid therein. Typically, thematerial overlying the opening can be thinner than the adjacentmaterial. Alternatively, or in addition, the weakened material can beformed by leaving this material relatively unsupported as compared tothe surrounding material of the lower wall.

While reservoirs have been described as having a sealed spaced formed inpart by a wall of the sealed space, other configurations are possible.For example, referring to FIG. 20A, a reservoir includes a plunger-likeactuation mechanism (e.g., a piercing member 342) and a gasket-likesealed space 343 having upper and lower layers 344, 345 respectively(e.g., upper and lower laminate layers). Liquid can be sealed betweenthe upper and lower layers. The sealed space can be surrounded by asupporting structure 346 (e.g., a toroidal gasket) that supports thesealed space at its upper and lower peripheral surfaces.

Referring to FIG. 20B, piercing member 342 is shown as being depresseduntil the piercing member 342 has pierced both the upper and lowerlayers bringing the liquid into communication with the microfluidicnetwork. A vent 346 adjacent the plunger allows gas trapped between thepiercing member and the upper layer of the sealed space to escapewithout being forced into the microfluidic network.

Referring to FIG. 20C, piercing member 342 is shown as fully actuated. Aportion of the piercing member has displaced a corresponding volume ofliquid from the sealed space and introduced the predetermined volume ofliquid into the microfluidic cartridge.

While the reservoirs have been described as having a sealed space thatmay be stationary with respect to a piercing member, otherconfigurations are possible. For example, FIG. 21A illustrates areservoir having a sealed space 347 that can be secured with (e.g.,integral with) respect to an actuation mechanism having a movable member348 (e.g., a plunger) and a piercing member 349 supported by a piercingmember support 350 that can be stationary with respect to the sealedspace. Typically, the sealed space can be defined by a cavity within themovable member and a lower wall 351 that seals liquid within the sealedspace. Piercing member can be configured to rupture the lower wall whenthe movable member can be depressed. Piercing member support has a shapegenerally complementary to the cavity of the movable member. Piercingmember support includes a channel 352 connected to a microfluidicnetwork to allow fluid released from the enclosed space to enter themicrofluidic network.

Referring to FIG. 21B, the movable member has been depressed so that thepiercing member has just ruptured the lower layer of the sealed space.Referring to FIG. 21C, the reservoir has been fully depressed onto thepiercing member and piercing member support. The volume of fluiddisplaced from the reservoir generally corresponds to the volume of thepiercing member support that enters the enclosed space. A channel 353allows air displaced by the moveable member to exit.

While reservoirs have been described as having a piercing member thatcan be secured with respect to some portion of the reservoir, otherconfigurations are possible. For example, referring to FIG. 22, areservoir includes an actuation mechanism 354 (e.g., a piercing membersuch as a needle-like piercing member) that can be unsecured withrespect to the reservoir. A sealed space 355 of the reservoir can bedefined by an upper wall 356 and includes a channel 357 extendingthrough a portion of a substrate 361 in which a microfluidic network canbe defined. A lower wall 358 of the sealed space separates the sealedspace from a channel 359 of the microfluidic network. The piercingmember occupies the channel 357 of the sealed space so that the piercingtip 360 of the piercing member rests against the lower wall 358.Depressing the upper wall 356 of the reservoir drives the piercingmember 354 through the lower wall and forces liquid within the sealedspace into the microfluidic network.

As another example, FIGS. 23A and 23B illustrate a reservoir includingan actuation mechanism (e.g., a piercing member) that can initially besecured to an interior of an upper wall of the reservoir but separatesat least partially from the upper wall upon actuation of the reservoir.

As yet another example, FIGS. 24A and 24B illustrate a reservoirincluding a piercing member 364 that can initially be secured to aninterior 365 of an upper wall 366 of the reservoir but substantiallyseparates (e.g., completely separates) from the upper wall uponactuation of the reservoir.

While reservoirs have been described as having an enclosed space thatcan be fixed or otherwise integral with a portion of the reservoir,other configurations are possible. For example, referring to FIG. 25, areservoir includes a capsule-like enclosed space 367 defined by an outerwall 368. The outer wall can generally be formed of a material having alow vapor transmission rate. Reservoir also includes an actuationmechanism having a moveable member 369 with a piercing member 370 thatpierces the enclosed space to release liquid therein. The liquid passesalong a channel 372 leading to a microfluidic network. A channel 371allows gas (e.g., air) otherwise trapped by the movable member to exit.

While reservoirs have been described as generally overlying an inlet toa microfluidic network, other configurations are possible. For example,referring to FIG. 26, a reservoir includes an enclosed space 373 inwhich liquid can be stored and a connecting portion 374 connected to aninlet 376 of a microfluidic network. The enclosed space 373 andconnecting portion 374 can be separated by a rupturable seal 375 (e.g.,a weak seal). In general, the rupturable seal 375 prevents liquid orvapor from exiting the enclosed space. However, upon the application ofpressure to the liquid (e.g., by depressing a wall 377 of the enclosedspace), the rupturable seal 375 ruptures allowing the liquid to passthrough the weak seal to the connecting portion and into themicrofluidic network 378.

A still further embodiment of a reservoir with a piercing member isshown in FIG. 27A, which shows a reservoir 2701 having an outer shell2703 and a piercing element 2704 that can both be made of the same pieceof material. Such a combined shell and piercing element can be formedfrom many processes known to one of ordinary skill in the art.Especially preferred processes can be vacuum thermo-forming andinjection moulding. Piercing element 2704 can generally be conical inshape, with the apex adjacent to a membrane 2702; its apex preferablydoes not exceed 0.040″. The piercing element will puncture membrane 2702and release liquid from reservoir 2701 when the outer shell can bedepressed. Representative dimensions are shown on FIG. 27A. Thereservoir may be constructed so that the upper surface can be level,with a flat protective piece 2705 covering the base of the conical shapeof piercing element 2704.

Yet another embodiment of a reservoir with a piercing member is shown inFIG. 27B, showing a reservoir 2711 having a single-piece outer shell2712 and piercing element 2714. Such a combined shell and piercingelement can be formed from many processes known to one of ordinary skillin the art. Especially preferred processes can be vacuum thermo-formingand injection moulding. Piercing element 2714 can be frustoconical inshape, with its narrower side adjacent to membrane 2713. Alternatively,piercing element 2714 can comprise several separate piercing elements,arranged within a conical space. Preferably there can be four suchpiercing elements where multiple elements can be present.

It is to be understood that the dimensions of the reservoir, piercingelement, shell and moulding shown in FIGS. 27A and 27B as decimalquantities in inches are exemplary. In particular, the dimensions can besuch that the shell does not collapse under its own weight and istypically not so as strong to prohibit depression of the piercing memberwhen required during operation of the cartridge.

Furthermore, the materials of the various embodiments can also be chosenso that the cartridge has a shelf-life of about a year. By this it ismeant that the thickness of the various materials can be such that theyresist loss, through means such as diffusion, of 10% of the liquidvolume contained therein over a desired shelf-life period.

Preferably the volume of the reservoir can be around 150 μl before ashell is depressed. Upon depression of a shell, the volume canpreferably be deformed to around half its original volume.

It is to be noted that completely filling the blister pack with a liquidreagent—with no remaining space for an air bubble results in a blisterthat requires application of significantly greater force than ispreferable. Accordingly, the blister(s) are typically filled to about80-95% of their volume, thereby reserving about 5-20%, typically 10-15%of the volume for air. Thus, in one embodiment, a blister that has atotal volume of 200 μl is filled with 170 μl of liquid.

Lysing Chamber

Exemplary lysing chamber 302, as shown in FIGS. 14A and 14B, is shown ina tower configuration, protruding from a plane of microfluidic cartridge300. Lysing chamber 302 can be divided into a primary lysing chamber 306and a waste chamber 308. In one embodiment, primary and waste chambers306, 308 are separated from one another so that material cannot passfrom one of the chambers into the other chamber without passing throughat least a portion of network 304. Primary lysing chamber 306 includes asample input port SP1 for introducing sample to chamber 306, a sampleoutput port SP2 connecting chamber 306 to network 304, and lyophilizedreagent LP that interact with sample material within chamber 306 asdescribed herein. Port SP2 is shown in FIG. 14A as being in the bottomof chamber 302. FIG. 15B shows a position of SP2 in relation to the restof microfluidic network 304. Input port SP1 includes a one way valvethat permits material (e.g., sample material and gas) to enter chamber306 but limits (e.g., prevents) material from exiting chamber 308 byport SP1. Typically, port SP1 includes a fitting (e.g., a Luer fitting)configured to mate with a sample input device (e.g., a syringe) to forma gas-tight seal. Primary chamber 306 typically has a volume of about 5milliliters or less (e.g., about 4 milliliters or less). Prior to use,primary chamber 306 can typically be filled with a gas (e.g., air).

Waste chamber 308 includes a waste portion W6 by which liquid can enterchamber 308 from network 304 and a vent 310 by which gas displaced byliquid entering chamber 308 can exit.

Lysing Reagent Particles

Lyophilized reagent particles LP of lysing chamber 302 include one ormore compounds (e.g., reagents) configured to release polynucleotidesfrom cells (e.g., by lysing the cells). For example, particles LP caninclude one or more enzymes configured to reduce (e.g., denature)proteins (e.g., proteinases, proteases (e.g., pronase), trypsin,proteinase K, phage lytic enzymes (e.g., PlyGBS)), lysozymes (e.g., amodified lysozyme such as ReadyLyse), cell specific enzymes (e.g.,mutanolysin for lysing group B streptococci)).

In some embodiments, particles LP alternatively or additionally includecomponents for retaining polynucleotides as compared to inhibitors. Forexample, particles LP can include multiple particles 218 surfacemodified with ligands as described above for processing chamber ofcartridge 200. Particles LP can include enzymes that reducepolynucleotides that might compete with a polynucleotide to bedetermined for binding sites on the surface modified particles. Forexample, to reduce RNA that might compete with DNA to be determined,particles LP may include an enzyme such as an RNAase (e.g., RNAseA ISCBioExpress (Amresco)).

In an exemplary embodiment, particles LP include a cryoprotecant,particles modified with ligands configured to retain polynucleotides ascompared to inhibitors, and one or more enzymes.

Typically, particles LP have an average volume of about 35 microlitersor less (e.g., about 27.5 microliters or less, about 25 microliters orless, about 20 microliters or less). In some embodiments, the particlesLP have an average diameter of about 8 mm or less (e.g., about 5 mm orless, about 4 mm or less) In an exemplary embodiment the lyophilizedparticle(s) have an average volume of about 20 microliters and anaverage diameter of about 3.5 mm.

Particles LP can be prepared as desired. Typically, the particles can beprepared using a cryoprotectant and chilled hydrophobic surface asdescribed hereinabove for other reagent particles. For example, asolution for preparing particles LP can be prepared by combining acryoprotectant (e.g., 6 grams of trehalose), a plurality of particlesmodified with ligands (e.g., about 2 milliliters of a suspension ofcarboxylate modified particles with poly-D-lysine ligands), a protease(e.g., 400 milligrams of pronase), an RNAase (e.g., 30 milligrams ofRNAseA (activity of 120 U per milligram), an enzyme that digestspeptidoglycan (e.g., ReadyLyse (e.g., 160 microliters of a 30,000 U permicroliter solution of ReadyLyse)), a cell specific enzyme (e.g.,mutanolysin (e.g., 200 microliters of a 50 U per microliter solution ofmutanolysin), and a solvent (e.g., water) to make about 20 milliters.About 1,000 aliquots of about 20 microliters each of this solution canbe frozen and desolvated as described above to make 1,000 pellets. Whenreconstituted, the pellets can typically be used to make a total ofabout 200 milliliters of solution.

Exemplary Operation of Microfluidic Cartridge

In use, various components of cartridge 300 can be operated as follows.Valves Vi and Vi′ of network 304 can be configured in the open state.Gates Gi and mixing gates MGi of network 304 can be configured in theclosed state. Reagent ports R1-R4 can be depressed, e.g., by applicationof mechanical force, to introduce liquid reagents into network 304, asdescribed hereinabove. A sample can be introduced to lysing chamber 302via port SP1 and combined with lyophilized particles LP within primarylysing chamber 306. Typically, the sample includes a combination ofparticles (e.g., cells) and a buffer solution. For example, an exemplarysample includes about 2 parts whole blood to 3 about parts buffersolution (e.g., a solution of 20 mM Tris at pH 8.0, 1 mM EDTA, and 1%SDS). Another exemplary sample includes group B streptococci and abuffer solution (e.g., a solution of 20 mM Tris at pH 8.0, 1 mM EDTA,and 1% Triton X-100).

In general, the volume of sample introduced can be smaller than thetotal volume of primary lysing chamber 306. For example, the volume ofsample may be about 50% or less (e.g., about 35% or less, about 30% orless) of the total volume of chamber 306. A typical sample has a volumeof about 3 milliliters or less (e.g., about 1.5 milliliters or less). Avolume of gas (e.g., air) can generally be introduced to primary chamber306 along with the sample. Typically, the volume of gas introduced canbe about 50% or less (e.g., about 35% or less, about 30% or less) of thetotal volume of chamber 306. The volume of sample and gas combine topressurize the gas already present within chamber 306. Valve 307 of portSP1 prevents gas from exiting chamber 306. Because gates G3, G4, G8, andG10 can be in the closed state, the pressurized sample can be preventedfrom entering network 304 via port SP2.

The sample dissolves particles LP in chamber 306. Reconstituted lysingreagents (e.g., ReadyLyse, mutanolysin) begin to lyse cells of thesample releasing polynucleotides. Other reagents (e.g., protease enzymessuch as pronase) begin to reduce or denature inhibitors (e.g., proteins)within the sample. Polynucleotides from the sample begin to associatewith (e.g., bind to) ligands of particles 218 released from particlesLP. Typically, the sample within chamber 306 can be heated (e.g., to atleast about 50° C., to at least about 60° C.) for a period of time(e.g., for about 15 minutes or less, about 10 minutes or less, about 7minutes or less) while lysing occurs. In some embodiments, opticalenergy can be used at least in part to heat contents of lysing chamber306. For example, the operating system used to operate cartridge 300 caninclude a light source 399 (e.g., a lamp primarily emitting light in theinfrared) disposed in thermal and/or optical contact with chamber 306.Such a light source can be that shown in connection with heater module2020, FIG. 7, reference numeral 2046. Chamber 306 includes a temperaturesensor TS used to monitor the temperature of the sample within chamber306. The lamp output can be increased or decreased based on thetemperature determined with sensor TS.

Continuing with the operation of cartridge 300, G2 can be actuated(e.g., opened) providing a path between port SP2 of primary lysingchamber 306 and port W6 of lysing waste chamber 308. The path extendsalong channel C9, channel C8, through processing region B1, and channelC11. Pressure within chamber 306 drives the lysed sample material(containing lysate, polynucleotides bound to particles 218, and othersample components) along the pathway. Particles 218 (withpolynucleotides) can be retained within processing region B1 (e.g., by afilter) while the liquid and other components of the sample flow intowaste chamber 308. After a period of time (e.g., between about 2 andabout 5 minutes), the pressure in lysing chamber 306 can be vented byopening gate G1 to create a second pathway between ports SP2 and W6.Double valves V1′ and V8′ can be closed to isolate lysing chamber 302from network 304.

Operation of cartridge 300 continues by actuating pump P1 and openinggates G2,G3 and G9. Pump P1 drives wash liquid in channel C2 downstreamof junction J1 through processing region B1 and into waste chamber W5.The wash liquid removes inhibitors and other compounds not retained byparticles 218 from processing region B1. When the trailing edge of thewash liquid (e.g., the upstream interface) passes hydrophobic vent H14,the pressure from actuator P1 vents from network 3Q4, stopping furthermotion of the liquid. Double valves V2′ and V9′ can be closed.

Operation continues by actuating pump P2 and opening gates G6, G4 and G8to move release liquid from reagent reservoir R1 into processing regionB1 and into contact with particles 218. Air vent AV1 vents pressureahead of the moving release liquid. Hydrophobic vent H6 vents pressurebehind the trailing edge of the release liquid stopping further motionof the release liquid. Double valves V6′ and V10′ can be closed.

Operation continues by heating processing region B1 (e.g., by heatingparticles 218) to release the polynucleotides from particles 218. Theparticles can be heated as described above for cartridge 200. Typically,the release liquid includes about 15 mM hydroxide (e.g., NaOH solution)and the particles can be heated to about 70° C. for about 2 minutes torelease the polynucleotides from the particles 218.

Operation continues by actuating pump P3 and opening gates G5 and G10 tomove release liquid from process region B1 downstream. Air vent AV2vents gas pressure downstream of the release liquid allowing the liquidto move into channel C16. Hydrophobic vent H8 vents pressure fromupstream of the release liquid stopping further movement. Double valveV11′ and valve V14 can be closed.

Referring to FIGS. 19A-19D, mixing gate MG11 can be used to mix aportion of release liquid including polynucleotides released fromparticles 218 and neutralization buffer from reagent reservoir R3. FIG.19A shows the mixing gate MG11 region prior to depressing reagentreservoir R3 to introduce the neutralization buffer into network 304.FIG. 19B shows the mixing gate MG11 region, after the neutralizationbuffer has been introduced into channels C13 and C12. Double valve V13′can be closed to isolate network 304 from reagent reservoir R3. Doublevalve V12′ can be closed to isolate network 304 from waste chamber W3.The neutralization buffer contacts one side of a mass 324 of TRS of gateMG11.

FIG. 19C shows the mixing gate MG11 region after release liquid has beenmoved into channel C16. The dimensions of microfluidic network 304(e.g., the channel dimensions and the position of hydrophobic vent H8)can be configured so that the portion of release liquid positionedbetween junctions J3 and J4 of channels C16 and C14 correspondsapproximately to the volume of liquid in contact with particles 218during the release step. In some embodiments, the volume of liquidpositioned between junctions J3 and J4 can be less than about 5microliters (e.g., about 4 microliters or less, about 2.5 microliters orless). In an exemplary embodiment, the volume of release liquid betweenjunctions J3 and J4 can be about 1.75 microliters. Typically, the liquidbetween junctions J3 and J4 includes at least about 50% ofpolynucleotides (at least about 75%, at least about 85%, at least about90%) of the polynucleotides present in the sample that enteredprocessing region B1. Valve V14 can be closed to isolate network 304from air vent AV2.

Before actuating mixing gate MG11, the release liquid at junction J4 andthe neutralization buffer at a junction J6 between channels C13 and C12can be separated by mass 324 of TRS (e.g., the liquids are not typicallyspaced apart by a volume of gas). To combine the release liquid andneutralization buffer, pump P4 and gates G12, G13, and MG11 can beactuated. Pump P4 drives the volume of neutralization liquid betweenjunctions J5 and J6 and the volume of release liquid between junctionsJ4 and J3 into mixing channel C15 (FIG. 19D). Mass 324 of TRS typicallydisperses and/or melts allowing the two liquids to combine. The combinedliquids include a downstream interface 335 (formed by junction J3) andan upstream interface (formed by junction J5). The presence of theseinterfaces allows more efficient mixing (e.g., recirculation of thecombined liquid) than if the interfaces were not present. As seen inFIG. 19D, mixing typically begins near the interface between the twoliquids. Mixing channel C15 can typically be at least about as long(e.g., at least about twice as long) as a total length of the combinedliquids within the channel.

The volume of neutralization buffer combined with the release liquid canbe determined by the channel dimensions between junction J5 and J6.Typically, the volume of combined neutralization liquid can be about thesame as the volume of combined release liquid. In some embodiments, thevolume of liquid positioned between junctions J5 and J6 can be less thanabout 5 microliters (e.g., about 4 microliters or less, about 2.5microliters or less). In an exemplary embodiment the volume of releaseliquid between junctions J5 and J6 can be about 2.25 microliters (e.g.,the total volume of release liquid and neutralization buffer can beabout 4 microliters).

Returning to FIGS. 15A, 15B, the combined release liquid andneutralization buffer move along mixing channel C15 and into channel C32(vented downstream by air vent AV8). Motion continues until the upstreaminterface of the combined liquids passes hydrophobic vent H11, whichvents pressure from actuator P4 stopping further motion of the combinedliquids.

Continuing with operation of cartridge 300, actuator P5 and gates G14,G15 and G17 can be actuated to dissolve the lyophilized PCR particlepresent in second processing region B2 in water from reagent reservoirR4. Hydrophobic vent H10 vents pressure from actuator P5 upstream of thewater stopping further motion. Dissolution of a PCR-reagent pellettypically occurs in about 2 minutes or less (e.g., in about 1 minute orless). Valve V17 can be closed.

Continuing with operation of cartridge 300, actuator P6 and gate G16 canbe actuated to drive the dissolved compounds of the lyophilized particlefrom processing region B2 into channel C31, where the dissolved reagentsmix to form a homogenous dissolved lyophilized particle solution.Actuator P6 moves the solution into channels C35 and C33 (venteddownstream by air vent AV5). Hydrophobic vent H9 vents pressuregenerated by actuator P6 upstream of the solution stopping furthermotion. Valves V18, V19, V20′, and V22′ can be closed.

Continuing with operation of cartridge 300, actuator P7 and gates G18,MG20 and G22 can be actuated to combine (e.g., mix) a portion ofneutralized release liquid in channel 32 between gate MG20 and gate G22and a portion of the dissolved lyophilized particle solution in channelC35 between gate G18 and MG20. The combined liquids travel along amixing channel C37 and into detection region D2. An air vent AV3 ventsgas pressure downstream of the combined liquids. When the upstreaminterface of the combined liquids passes hydrophobic vent H13, thepressure from actuator P7 can be vented and the combined liquids can bepositioned within detection region D2.

Actuator P8 and gates MG2, G23, and G19 can be actuated to combine aportion of water from reagent reservoir R4 between MG2 and gate G23 witha second portion of the dissolved lyophilized particle solution inchannel C33 between gate G19 and MG2. The combined liquids travel alonga mixing channel C41 and into detection region D1. An air vent AV4 ventsgas pressure downstream of the combined liquids. When the upstreaminterface of the combined liquids passes hydrophobic vent H12, thepressure from actuator P8 can be vented and the combined liquids can bepositioned within detection region D1.

Continuing with operation of cartridge 300, double valves V26′ and V27′can be closed to isolate detection region D1 from network 304 and doublevalves V24′ and V25′ can be closed to isolate detection region D2 fromnetwork 304. The contents of each detection region (neutralized releaseliquid with sample polynucleotides in detection region D2 with PCRreagents from dissolved lyophilized particle solution and deionizedwater with PCR reagents from dissolved lyophilized particle solution indetection region D1) can be subjected to heating and cooling steps toamplify polynucleotides (if present in detection region D2). The doublevalves of each detection region prevent evaporation of the detectionregion contents during heating. The amplified polynucleotides cantypically be detected using fluorescence detection. Thus, typicallyabove one or both of detection regions D1, D2, is a window (as in, e.g.,FIG. 9) that permits detection of fluorescence from a fluorescentsubstance in reaction mixture when a detector is situated above thewindow.

While cartridges for carrying out various stages of processing sampleshave been shown and described herein as having a generally planarconfiguration, other configurations can be used and are consistent withan integrated system as described herein. For example, a cartridgehaving a generally tube-like or vial-like configuration is described inU.S. patent application publication no. 2006-0166233, incorporatedherein by reference.

EXAMPLES

The following Examples are illustrative and are not intended to belimiting.

Example 1: Apparatus for Polynucleotide Processing

This non-limiting example describes various exemplary embodiments of anapparatus, system, microfluidic cartridge, kit, methods, and computerprogram product, as shown in FIGS. 28-40.

For example, FIG. 28 is a diagram of an apparatus 800 that can operateas a small bench-top, real time, polynucleotide analysis system. Such asystem can perform a variety of tests on polynucleotides, for example,testing samples from patients for signatures of one or more infectiousdiseases. The apparatus can function, for example, as a real timepolynucleotide analysis system for use in the clinical diagnostic marketto assist clinical personnel in testing and treating patients beforethey leave the medical environment. Apparatus 800 can include, forexample, a housing having a display output 802, a lid 804 having ahandle 805, and a bar code reader 806. Referring to FIGS. 28 and 36,apparatus 800 can also include a receiving bay 807, which can be coveredby lid 804. In various embodiments, apparatus 800 can be portable, forexample, apparatus 800 can weigh about 10 kg and can have dimensions ofapproximately 25 cm wide by 40 cm deep by 33 cm high.

Apparatus 800 can be used with a sample kit 810, shown in FIG. 29. Thesample kit 810 can include, for example, a microfluidic cartridge 812with an optional label 813 (e.g., a barcode label), a sample container814 with an optional label 815 (e.g., a barcode label), an optionalfilter 818, an optional pipette tip 820, and an optional syringe 822.Referring to FIG. 30, one or more components of the sample kit 810(e.g., microfluidic cartridge 812) can be packaged, e.g., in a sealedpouch 824 which can optionally be hermetically sealed with an inert gassuch as argon or nitrogen.

Microfluidic cartridge 812, as depicted in FIG. 31, can include a sampleinlet 826, a plurality of self-pierceable reservoirs 828, a lysisreservoir 830, a waste reservoir 832, an optional label 813 (e.g., a barcode), and a registration member 836 (e.g., a beveled corner). Referringto FIGS. 31 and 36, registration member 836 can fit a complementaryregistration member feature 809 of receiving bay 807 in apparatus 800,which can be used to facilitate orientation of cartridge 812 wheninserted in the apparatus 800. In some examples, microfluidic cartridge812 can be designed to be slightly smaller (e.g., 50-300 microns,typically 200-300 microns) than well 807 in heater/sensor module 842 tofacilitate placement and removal of microfluidic cartridge 812.

Referring to FIGS. 32 & 33, the labels, e.g., bar codes 813 and 815, onthe microfluidic cartridge 812 and/or sample container 814 can berecorded by apparatus 800, typically before performing a test, e.g., byentering a label code manually or by using a bar code reader 806.

In preparation for testing a sample, pipette tip 820 from kit 810 may beattached to the syringe 822, and a sample may be drawn from samplecontainer 814 into syringe 822. Referring to FIG. 34, filter 818 canthen be attached to lysis reservoir 830 of microfluidic cartridge 812via sample inlet 826 (e.g., using a luer lock with a duckbill valve atsample inlet 826) and the contents (e.g., a sample/air mixture) ofsyringe 822 may be injected into microfluidic cartridge 812 throughfilter 818. Additional air (e.g., 1-3 mL) can be drawn into syringe 822(as shown in FIG. 35) so that microfluidic cartridge 812 can bepressurized (e.g., 5-50 pounds per square inch (psi) with respect toambient pressure, typically between 5-25 psi, more typically between10-15 psi, with respect to ambient pressure). Microfluidic cartridge 812can contain buffers, reagents, and the like, e.g., in lysis reservoir830 in the form of liquids, solids, lyophilized reagent pellets, and thelike. Microfluidic cartridge 812 can be agitated to mix the injectedsample with the buffers, reagents, etc.

Referring to FIG. 35, microfluidic cartridge 812 can be pressurizedusing syringe 822 and filter 818 with added air. Microfluidic cartridge812 can be placed in receiving bay 807 of apparatus 800, as shown inFIG. 36, and can be seated in a single orientation in receiving bay 807of apparatus 800 due to the interaction between registration member 836on microfluidic cartridge 812 and complementary registration member 809in receiving bay 807.

As shown in FIGS. 37 and 38, closure of lid 804 of apparatus 800 canserve to block ambient light from the sample bay. Additionally, closureof lid 804 can place an optical detector contained in lid 804 intoposition with respect to microfluidic cartridge 812. Also, lid 804 ofthe apparatus can be closed to apply pressure to microfluidic cartridge812 to ensure thermal contact in well 807 with heating/sensor module842. Referring to FIGS. 39 & 40, heating/sensor module 842 of apparatus800 can be removable for cleaning, maintenance, or for replacement witha custom heating stage for a particular microfluidic cartridge 812.

Example 2: Apparatus for Polynucleotide Processing

This non-limiting example describes various embodiments of an apparatus,system, microfluidic cartridge, kit, methods, and computer programproduct, in particular, various aspects of using apparatus 800 asdescribed in Example 1 relating to exemplary aspects of the method andthe computer program product.

Referring to FIGS. 28-40, apparatus 800 may include or be configuredwith a computer program product in the form of control software. Thesoftware can provide apparatus with a “READY MODE” (e.g., standby mode)when not being used for analysis where the apparatus 800 can be pluggedin and can await user input. An operator may slide open the lid 804using the handle 805 to its full open position. The software andapparatus 800 may be configured to indicate on display output 802 thatlid 804 is open and apparatus 800 is ready. The software and apparatus800 may be configured to to perform a hardware and/or software selftest. The software and apparatus 800 may be configured to request entryof a user ID and password screen, for example, to allow a user to log inby using a touch screen interface (e.g., by touching keys of an emulatedkeyboard on the display output 802), by scanning in a bar coderepresenting the user, such as found on an ID badge, with bar codereader 806, and the like.

A user may then remove the microfluidic cartridge 812 and the samplecontainer 814 from the sealed pouch 824. The software and apparatus 800may be configured to request that the bar code 813 on the microfluidiccartridge 812 and/or the bar code 815 on the sample container 814 bescanned in using the bar code reader 806, as in FIGS. 32 and 33, beforeperforming any test. The software and apparatus 800 may be configured toperform qualifying tests based on the bar codes (e.g., to determinewhether microfluidic cartridge 812 and sample container 814 came fromthe same sealed pouch 824, whether kit 810 has passed an expirationdate, whether kit 810 can be configured to be used with a particularanalysis sequence to be conducted by the software and apparatus 800 andthe like) before performing nucleic acid tests. The display output 802may then advance to a screen requiring an input, such as a patientidentifier. Apparatus 800 may also allow the patient information to beentered by scanning a bar code (e.g., on a medical ID bracelet assignedto the patient) using the bar code reader 806. Display output 802 canprovide information to the user regarding, for example, results of atest that was previously run, selection options for tests to be run, andthe like. Examples of information that may be provided include, but arenot limited to: a test determination (e.g., positive/negative result),an internal control result (e.g., positive and/or negative), and/orpatient results. In this example, the user may also be prompted to allowthe apparatus 800 to perform additional tasks with the test data, suchas recording or outputting the data to a printer, storage device, or toa computer, and the like.

Various embodiments of the software and apparatus 800 may be configuredto allow a user to perform one or more optional functions (e.g.,adjustments to apparatus 800 or the software) including, but not limitedto: modifying user settings, modifying logout settings, setting thesystem clock, modifying display settings, modifying QC requirements,setting report preferences, configuring an attached printer, configuringa network connection, sending data via a network connection, selectingor adapting data analysis protocols, or the like.

In various embodiments, the software can include a user interface anddevice firmware. The user interface software can allow for aspects ofinteraction with the user including, but not limited to, enteringpatient/sample information, monitoring test progress, error warnings,printing test results, uploading of results to databases, updatingsoftware, and the like. The device firmware can operate apparatus 800during the analytical tests and can have a generic portion that can betest independent and a portion specific to the test being performed. Thetest specific portion (“protocol”) can specify the microfluidicoperations and their order to accomplish the test. FIG. 41A shows ascreen capture from an exemplary interface displaying real time heatsensor data. FIG. 41B shows a screen captures from an exemplaryinterface displaying real time optical detector data.

Example 3: Apparatus for Polynucleotide Processing

This non-limiting example describes various embodiments of the claimedapparatus, system, microfluidic cartridge, kit, methods, and computerprogram product. In one embodiment, the apparatus 800, shown in FIG. 28,may be a self-contained, real-time PCR device based on microfluidictechnology for rapid and accurate diagnosis of pathogens (e.g., Group BStreptococcus (GBS) colonization in prenatal women). In an exemplaryembodiment, when the microfluidic cartridge 812 (FIG. 31) can beinstalled, the apparatus 800 may actuate on-cartridge operations, detect& analyze the products of a PCR amplification and/or display the resultson a graphical user interface. The microfluidic cartridge 812 mayinclude a plurality of chambers and/or subunits, for performing avariety of tasks, with limited or no intervention by the user. FIG. 42is a schematic representation of the various chambers and/or subunits inan exemplary microfluidic cartridge 812. Microfluidic cartridge 812 canaccept unprocessed clinical samples (e.g., vaginal/rectal swab dipped intransport buffer in the case of GBS) via the sample inlet 826, which maybe a luer-style injection port. Clinical swab samples containing humanand bacterial cells and debris can be routinely collected in 2 ml oftransport buffer. However, small volumes (e.g., on the order of a fewmicroliters) can be easily processed on microfluidic devices. Theincorporation of an interface between macro and micro operations canallow the adaptation of microfluidic technology for clinical diagnosis.Upon injection of a sample, the cartridge may be placed in the apparatus800 and further operations; for example, sample preparation, reagentmetering/mixing, and PCR amplification/detection may be performed in anautomated and hands-free manner.

FIG. 43 is a schematic representation of the steps relating to PCR anddetection that can be performed in an exemplary microfluidic cartridge812. In various embodiments, the steps to process samples for PCR baseddetection & diagnosis of pathogens (e.g., GBS from vaginal/rectal swabs)may include: lysis to release DNA (e.g., lysis of GBS cells to releasepolynucleotides), capture and concentration of DNA, and minimization ofinhibitors and competitors to clean-up the DNA for PCR compatibility.Inhibitors present in clinical samples can increase the risk of a falsenegative result for PCR based diagnostic tests unless their influencecan be mitigated through sample clean-up.

Cell lysis can be achieved by methods known to the art, for example,heat and/or chemical activation. In some embodiments, after a 1.0+/−0.2mL sample can be injected into the lysis reservoir 830 via the sampleinlet 826, the apparatus 800 may cause the sample to be mixed with lysisreagents from the wet reagent storage 838 and heated (e.g., for 7minutes) in the lysis reservoir 830. Using this protocol, greater than90% lysis efficiency has been achieved for GBS and other bacterialcells. The lysis reagents may also incorporate a cationic polyamidemodified polycarbonate-polystyrene latex bead based DNA affinity matrix(e.g., retention member 821) to capture the negatively charged DNA whichmay be released during the lytic process. The affinity beads can bind tonegatively charged DNA with very high affinity while potential PCRinhibitors can either fail to bind or can be removed during subsequentwash steps.

In one exemplary embodiment, the apparatus 800 may also automate thecapture and cleaning of DNA from crude sample lysate (e.g., GBS samplelysate) to generate “PCR-ready” DNA. The contents of the lysis reservoir830 (e.g., 1.0+/−0.2 mL sample and reagents) may be transferred to theDNA processing chamber 840. Affinity beads with bound DNA from the inputsample can be trapped using an in-line bead column (e.g., a filter withspecific pore size) and an on-cartridge pump can be used to wash theaffinity beads to remove non-specifically bound moieties as well assoluble inhibitors by performing a buffer exchange. The bound DNA may bereleased by known methods, for example, by heating the affinity beads(e.g., to 80° C.) and/or by using a release buffer. The intact DNA canbe recovered with this single step release in very small volume (3-4 μl)thereby achieving a significant concentration of the original targetDNA. Other methods known to the art can be employed by the system toachieve cell lysis and DNA capture, wash, and release.

In the example described here, the basis for the real-time PCR assayused is the TaqMan® assay, the schematic operation of as depicted inFIG. 44. However, other assay techniques known to the art may be used(e.g., SYBR-Green I fluorescence). The TaqMan® PCR reaction exploits the5′ nuclease activity of certain DNA Polymerases to cleave a TaqMan®probe during PCR. The TaqMan® probe contains a reporter dye at the5′-end of the probe and a quencher dye at the 3′-end of the probe.During the reaction, cleavage of the probe can separate the reporter dyeand the quencher dye, which may result in increased fluorescence of thereporter. Accumulation of PCR products can be detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe is intact, the proximity of the reporter dye to the quencher dyecan result in suppression of the reporter (Fluorescence emission can beby Förster-type energy transfer). During PCR, if the target of interestcan be present, the probe can anneal between the forward and reverseprimer sites. The DNA Polymerase typically can cleave the probe betweenthe reporter and the quencher if the probe hybridizes to the target. Theprobe fragments can then be displaced from the target, andpolymerization of the strand can continue. The 3′-end of the probe canbe blocked to prevent extension of the probe during PCR.

This process can typically occur, for example, in every thermal cycleand should not interfere with the exponential accumulation of product.The increase in fluorescence signal can typically be detected if thetarget sequence can be complementary to the probe and can be amplifiedduring PCR. TaqMan® assay can offer a two-fold stringency (the primertypically binds and the probe typically binds to the target sequence)and hence detection of any nonspecific amplification can be reduced oreliminated.

Real-time PCR primers and probe sets for GBS (Streptococcus agalactiae)have been designed & tested using clinical samples. The PCR reagents mayinclude a pair of hybridization primers specific to the portion of thecfb gene between positions 328 and 451 encoding the CAMP factor(Christie, Atkins and Munch-Petersen, see, e.g., Boll Ist SieroterMilan, (1955 July-August); 34(7-8):441-52). The CAMP factor is adiffusible extracellular protein and is produced by the majority of GBS.The gene encoding CAMP factor, cfb gene (GenBank access number: X72754),can be present in GBS isolate and has been used for the development of aPCR based identification of GBS (Danbing K., et al., (2000), ClinicalChemistry, 46, 324-331). Further, a specific TaqMan® style fluorogenicprobe has also been designed & tested, in one example, to recognize thesequence amplified between the primers allowing real-time detection byusing fluorescence measurements.

In order to evaluate the DNA clean-up process and monitor theperformance of our primers at run-time, positive internal controlplasmids (e.g., as depicted in FIG. 45) can be employed. In variousembodiments, the specific GBS primers have been employed to constructinternal control plasmids as follows. A piece of random DNA sequenceflanked by the specific PCR primers was generated by oligonucleotidesynthesis. Any possible homology between this sequence and other DNAsequences especially Strep. agalactiae DNA, available in Genbank waschecked carefully. A fluorogenic TaqMan® style probe was designed torecognize the amplicons generated from this sequence and a fluorophore(Cal Orange 560 or analog) different from the one used for GBS targetsequence DNA (FAM or analog) was used for simultaneous dual colorfluorescent detection. In certain embodiments, the amount of theinternal control plasmid to be included in the PCR reaction wasoptimized to permit amplification of the internal control productwithout significant detrimental effect on the GBS specificamplification. In some examples, the specificity of the probes forinternal controls was also tested and optimized by PCR using the DNApurified from the pathogens included in the cross reactivity testinglist specified previously and GBS DNA.

In an exemplary embodiment, a robust system for carrying out rapidthermo-cycling using a microfluidic volume was designed, developed, andimplemented in a microfluidic format. The microfluidic volumes that canbe accommodated range from about 0.01 μl to about 10 μl, wherein theprincipal limitation on the lower limit is sensitivity of detection.Exemplary volumes are in the range 0.5-4.5 μl. Still other exemplaryvolumes are 2 μl. FIGS. 41A and 41B show screen shots of an exemplaryapparatus 800 output (e.g., as seen on the touch sensitive LCD 846).FIG. 41A shows a microfluidic PCR module undergoing rapid thermo-cyclingand FIG. 41B shows a real-time PCR assay. This data can be available tothe user of apparatus 800 or can be hidden. In this case, the GBScartridges were also designed to accommodate two PCR chambers to allowfor incorporating an on-board negative control with each sample run toimprove the fidelity of the result. In some examples, the chemistry hasbeen optimized and a compatible detection system developed to enabletwo-color multiplex PCR thereby facilitating the use of internalpositive controls to check for efficiency of sample prep and properperformance of the associated instrumentation. Due to very small thermalmasses and efficient feedback-control based algorithms, it can bepossible to perform ultra-fast thermo-cycling such that a typical50-cycle PCR can be completed in approximately 20 minutes. Heat requiredfor thermo-cycling can be provided by the heater/sensor module 842 andreal-time multiplexed detection can be carried out by the optics module(e.g., the fluorescent detection module 844).

In various embodiments, any number (e.g., 0, 1, 2, or all) of thereagents for performing the PCR can be incorporated on the cartridge ina lyophilized format. At the time of use, the lyophilized PCR reagentscan be reconstituted using, for example, deionized water, which may bestored on the microfluidic cartridge 812 in a blister format (e.g., in aself-pierceable reservoir 828). The reconstituted PCR reagents can bealiquoted into, e.g., two parts. In various embodiments, PCR ready DNA(output of the sample preparation module) can be mixed one aliquot andsent to the first PCR channel for real-time PCR (sample PCR). DI watercontaining non-target DNA can be mixed with the second aliquot of thePCR reagents and can be sent to the other PCR chamber (Negative PCR) toserve as the negative control.

Example 4: Apparatus for Polynucleotide Processing

In various embodiments, a microfluidic system (e.g., microfluidiccartridge 812) can include components such as micropumps formoving/mixing liquid drops, microreactors for performing thermallyinitiated biochemical reactions, and microvalves or microgates to enablecontrol of the liquid pumping operations as well as to isolate regionsof the cartridge such as the PCR chambers during thermocycling.

In some embodiments, a liquid drop handling system can be used toproduce liquid-sample injection and motion based on thermally actuatedpumps (e.g. thermo-pneumatic pumping) that may be operatedelectronically without the use of mechanical valves. For example, byheating air trapped inside chambers that can be connected to the mainchannel, significant air pressure can be generated for thermo-pneumaticpumping. Increasing the temperature of the air can cause the pressureinside the chamber to rise until the pressure can be high enough tosplit off a drop (meter an aliquot) and move it to the desired location.This technique can be implemented as an on-cartridge actuation mechanismand may use, for example, molded chambers, channels and heaters.Typically, this can avoid mechanical moving parts and can facilitatefabrication. FIG. 46 shows photos of a demonstration showing the mixingof two fluids (“A”—blue and “B”—orange) using the drop-handling systemdescribed above. Pressure pumps P1 and P2 can be activated in aprecisely controlled manner which can force the liquids to move asalternating rolling discrete drops along channel M, where they can mix,and finally be positioned into chamber C, where the PCR can take place.

In some embodiments, thermally expansive materials such as gas, readilyvaporizable liquid (e.g., vaporizable between 25° C. and 100° C. at 1atmosphere), and/or a thermally-expanding polymer (e.g., Expancel) maybe introduced in thermally actuated pumps (e.g., thermopneumatic airchambers), which may minimize the size of the pumps to generatedifferential pressures greater than 5 psi. These materials can expand,e.g., by over 100% when a threshold temperature can be reached, causingit to partially or completely fill up the thermopneumatic chambercausing further compression of the air.

For example, FIGS. 47A and 47B depict a thermally actuated pump 500based on a phase transition material (PTM) 510 (a PTM of a known meltingpoint (e.g., 60 C/75 C/90° C.) such as a paraffin wax, solder, Expancel,or the like), in a closed (FIG. 47A) and open (FIG. 47B) configurations.The PTM 510 can be constrained to a specific location. The specificlocation can be a sealed chamber 512 with the PTM 510 (typically about50-100 μl) deposited on a laminate. Upon heating the pump to atemperature above 120° C., the PTM 510 irreversibly expands (up to 40times its original size), compressing the air within the chamber 512,displacing the air from the chamber, and causing the adjacent gate 514to open. FIGS. 47C and 47D show another example of a pump 501 withexpancel polymer 511 in chamber 513 which can be actuated to operategate 515.

An exemplary clinical sample input into the microfluidic cartridge 812can have a volume of approximately 1 milliliter. After enzymatic/thermallysis of the cells, the released DNA can be bound toaffinity-microbeads. These microbeads can be, for example, on the orderof 10 microns in size. In various embodiments, a total amount of beadsin the range of a few million can be used per microfluidic cartridge 812for DNA concentration. In some cases, a minimum pressure of 10 psi(e.g., 10 psi, 11 psi, or 15 psi) may be used to concentrate the beadsagainst an inline-filter area of a few square millimeters (pore size of8 microns) in a few minutes (e.g. 3 minutes). This pressure can begenerated, for example, by injecting extra air (e.g., 1-3 mL) into thebulk lysis chamber of the microfluidic cartridge 812. In someembodiments, a one-way duckbill valve at the luer inlet can be used tominimize or prevent air pressure from escaping through the inlet.

Reagents which can be employed for sample preparation and PCR reactionscan be pumped into the microfluidic network by depressing the reagentblister domes by the slider of the instrument during use of theinstrument.

In exemplary embodiments, the enzymes typically employed for cell lysis,DNA capture, and for performing real-time PCR can be lyophilized intopellets and stored at different locations of the cartridge. The contactof air can be minimized by storing the pellets in the microfluidiccartridge 812, for example, in a nitrogen purged chamber or a in achannel structure sealed on either ends of the microchannel by thermalgates. Buffers typically employed for sample preparation, reagenthydration and PCR can be stored in hermetically sealed reagent blisters(e.g., the self-pierceable reservoirs 828). Materials used for makingthe reagent blister can have high moisture vapor barrier and canminimize the loss of liquid during storage of the cartridge over a year.Waste generated from the clinical sample as well as the various washbuffers can be stored on-board the cartridge in chambers andmicrochannels (e.g., the waste reservoir 832, which can typically beleak-resistant).

Example 5 Aspects of Real-Time PCR

A plurality of steps that can be used for the accurate, real-time PCRbased diagnosis of pathogens (e.g., Group B Streptococcus (GBS)colonization in prenatal women) was integrated into a single,microfluidic technology based, disposable cartridge, as shown in FIG.48. Exemplary steps which can be performed in a “sample in; result out”style operation on such a cartridge include: bulk lysis; DNA capture,wash, and release; and PCR preparation and execution. In variousembodiments of this cartridge, the sample and reagents can be containedon-board the microfluidic cartridge 812 (as shown in FIG. 31) and theretypically is no need for manual interaction with the operation except,for example, during the act of injecting the sample into the device.Strategically positioned hydrophobic vents can be included to removetrapped air formed during processing and reagents typically employed forthe assay can be packaged as lyophilized beads in the cartridge. Liquidsrequired for reconstitution can be stored in blister pouches thatrelease at the time of use.

In various embodiments, samples (e.g., GBS samples) can be introducedthrough the sample inlet 826, which may have a luer fitting foraccommodating a syringe. A pre-filter (e.g., attached to the syringe)can be used to remove at least a portion of crude impurities from thesample and, in some embodiments, the sample (e.g., 1 mL) can be lysed inthe lysis chamber using heat and/or lytic enzymes. Enzymes such aspronase, protienaseK, and RNAaseA can be used (e.g., during the lysisstep) to remove the inhibitory proteinaceous matter and competing RNAmolecules. Referring to FIG. 49, DNA capture beads can also be includedin the master mix. The lysed liquid sample (e.g., containing the GBSand/or other DNA bound to affinity beads) can be backflowed into themicrofluidic cartridge from the lysis chamber outlet. In some examples,the affinity beads with captured DNA can be retained by an in-linefilter while the unwanted debris and the excess liquid may be sent tothe waste chamber. In such embodiments, the beads used may benon-magnetic, or magnetic, and the filter is selected to discriminateparticles by size. In other embodiments, the beads are magnetic, and areconcentrated at a particular location of the microfluidic cartridge,such as a chamber or a channel, by applying a magnetic field configuredto concentrate lines of flux at the location in question. The magnetemployed may be an electromagnet, such as controlled by the processor toswitch on and off at specified times during sample analysis. The magnetmay alternatively be a permanent magnet, such as one that is moved intoand out of place when required.

In some embodiments, the waste chamber is equipped with an anti-foamingagent, such as simeticone. Vigorous bubble formation can occur in thewaste chamber because liquid enters it at high speed and, upon mixingwith air in the waste chamber, foams. It is undesirable if the foamoverflows from the waste chamber. Presence of a de-foaming agent canmitigate this phenomenon. The defoaming agent can be present in powder,tablet, pellet, or particle form.

In some embodiments, the beads can be subject to washing to removeunbound and non-specifically bound matter and the cleaned DNA can bereleased into a small volume (˜3 μl) compartment and concentrated (e.g.,by a factor of about 300). In various embodiments, the concentrated DNAcan then be mixed with the appropriate PCR reagents and/or be sent to aPCR channel for real-time PCR. An Internal Control plasmid (along withits cognate Probe) may also be included in the first PCR channel, whichcan act as a positive control. In a second PCR channel (if present), DIwater containing non-target DNA and the internal control can be mixedalong with the PCR reagents, to act as a negative control.

A user may introduce a sample in the bulk lysis sample throughluer-duckbill valve (e.g., the sample inlet 826), shake gently todissolve lysis reagent pellets, and introduce an excess amount of air(e.g., 0.25-0.75 ml) into the lysis chamber to over-pressurize the lysischamber. The absolute pressure (P) generated in the lysis chamber havinga chamber volume, V_(chamber), is related to the amount of liquid sampleinjected, V_(sample), and the volume of extra air injected,V_(extra air), by the formula:

$P = \frac{P_{atm}\left( {V_{Chamber} - V_{sample}} \right)}{\left( {V_{Chamber} + V_{sample} + V_{extraAir}} \right)}$

The microfluidic cartridge 812 then may be placed in the apparatus 800and the slider module 848 closed. On closing, the slider module 848 maypress reagent blisters (e.g., self-piercing reservoirs 828), causingthem to burst and release reagents (e.g., wash buffer, release buffer,neutralization buffer and water) into the channels with reagents.

In various embodiments, the apparatus 800 may perform any or all of thefollowing steps. In referring to FIGS. 50A-50J in the following, variouselements can also be located with the same designators in FIGS. 15A and15B. Referring to FIG. 50A, valves (V3, V4, V5, V7, V12, V13, V15, V16,V23) on either ends of the channel holding the 4 reagents may be closedand the bulk lysis lamp may be activated to heat the bulk lysis chamber,for example, to 60° C. for 7 minutes (e.g., using temperature sensor Lin FIG. 50B for feedback). Referring to FIG. 50B, Gate G1 can be openedto drain, for a predetermined amount of time (e.g., 2-5 minutesdepending on the sample type), the liquid (containing lysate, DNA boundto DNA-affinity beads, etc) through the bead capture filter into thewaste chamber. DNA-beads may be trapped against the inline filter whileother liquid flows to the waste chamber. Gate G7 can be opened to ventthe excess liquid and/or pressure in the lysis chamber into the wastechamber. Valves V1 and V8 may be closed to block the lysis chamber andthe waste chamber, respectively.

Referring to FIG. 50C, in some examples, trapped beads may be washed bypumping wash buffer through the bead column using pump E1 and openinggates G2, G3, and G9 to position wash buffer downstream of hydrophobicvent H2. The valves isolating the wash buffer channel (e.g., V2) and thewash buffer waste (e.g., V9) may be closed.

Referring to FIG. 50D, release buffer can be pumped, by using pump E2and opening gates G6, G4, and G8 to fill up a bead column and positionrelease buffer downstream of air vent H1. The valves blocking therelease buffer channel (e.g., V6) and channel downstream of the beadcolumn (e.g., V10) can be closed and the beads heated to, for example,70 C for 2 minutes which may release the DNA from the DNA-affinitybeads.

Referring to FIG. 50E, the released DNA can be pumped, using pump E3 andopening gates G5 and G10 to a position downstream of the hydrophobicvent H4, at which time the valves V11 and V14 can be closed. Referringto FIG. 50F, a portion of the released DNA (between junction G11 and a1)and portion of the neutralization buffer (between G11 and a2) can bemixed by using pump E4 and opening gates G12, G11, and G13. Mixing ofthe composite liquid plug between a1 and a2 may happen after it can bepumped through the neutralization mix channel and positioned downstreamof the hydrophobic vent H4. G11 may be a zero-dead volume gate thatbrings two liquid channels close to each other without trapping airbubbles during the merging of the two liquid plugs. The two valves (V21and V22) on the ends of the neutralized sample can be closed and, nowreferring to FIG. 50G, DI water may be pumped using pump E5 and openinggates G14, G15, and G17 to dissolve PCR-reagent pellet. The liquid maybe positioned using hydrophobic vent H12. After a period of time longenough for complete dissolution of the lyophilized PCR reagent pellet(e.g., approximately one minute) valve V17 may be closed.

Referring to FIG. 50H, dissolved enzyme can be pumped through the enzymemixing channel by using pump E6, opening gate G16, and using hydrophobicvent H5 to assist in placement of the fluid. In various embodiments, themixed enzyme may be distributed (e.g., in 2 equal parts) for multiplereactions to the sample PCR mix section as well as negative PCR mixsection, at which time, valves V18, V20, and V19 may be closed.

In various embodiments, referring to FIG. 50I, a portion of theneutralized DNA (between junction G20 and b1) and portion of the enzyme(between G20 and b2) may be mixed by using pump E7 and opening gatesG18, G20, and G22. Mixing of the composite liquid plug between a1 and a2can occur after it can be pumped through the neutralization mix channeland positioned downstream of the hydrophobic vent 116. A portion of theDI water (between junction G21 and c1) and a portion of the enzyme(between G21 and c2) can be mixed by using pump E8 and opening gatesG19, G21, and G23. Mixing of the composite liquid plug can occur betweenc1 and c2, after it can be pumped through the neutralization mix channeland positioned downstream of the hydrophobic vent H7.

Referring to FIG. 50J, PCR valves V24, 25, 26, 27, in some examples, canbe closed to perform sample PCR and Negative control PCR. In variousembodiments, light (e.g., fluorescence) can be detected using theoptical system in the slider. In some examples, the software candetermine the presence of target (e.g., GBS) in the sample based onfluorescence data and may report the results.

Example 6: Apparatus for Polynucleotide Processing

This non-limiting example shows CAD views of various exemplaryembodiments of the apparatus, system, microfluidic cartridge, kit,methods and computer program product, as further described herein.

FIG. 51 shows a side view of a lever assembly 1200, with lever 1210,gear unit 1212, and force member 1214. Assembly 1200 can be used toclose the lid of the apparatus and (through force members 1214) applyforce to a microfluidic cartridge 1216 in the receiving bay 1217. Oneforce member is visible in this cut away view, but any number, forexample four can be used. The force members can be, for example, amanual spring loaded actuator as shown, an automatic mechanicalactuator, a material with sufficient mechanical compliance and stiffness(e.g., a hard elastomeric plug), and the like. The force applied to themicrofluidic cartridge 1216 can result in a pressure at the surface ofthe microfluidic cartridge 1216 of at least about 0.7 psi to about 7 psi(between about 5 and about 50 kilopascals), or in some embodiments about2 psi (about 14 kilopascals.

FIG. 52 shows a side view of lever assembly 1200, with microfluidiccartridge 1216 in the receiving bay 1217. A heat pump 1219 (for example,a xenon bulb as shown) can function as a radiant heat source directed ata sample inlet reservoir 1218, where the heat can lyse cells inreservoir 1218. A thermally conductive, mechanically compliant layer1222 can lie at an interface between microfluidic cartridge 1216 andthermal stage 1224. Typically, microfluidic cartridge 1216 and thermalstage 1224 can be planar at their respective interface surfaces, e.g.,planar within about 100 microns, or more typically within about 25microns. Layer 1222 can improve thermal coupling between micro fluidiccartridge 1216 and thermal stage 1224. Optical detector elements 1220can be directed at the top surface of microfluidic cartridge 1216.

FIG. 53 shows a close-up of receiving bay 1217.

FIG. 54 shows a close-up of the interface between microfluidic cartridge1216, thermally conductive, mechanically compliant layer 1222, andthermal stage 1224.

FIG. 55 shows a top view of assembly 1200. In addition to mechanicalmembers 1214, guide members 1226 can be employed.

FIG. 56 is a close-up of FIG. 55.

FIGS. 57-59 are a series of pictures of lever 1210 in action. Shown inaddition in gear assembly 1212 can be cam 1228, which enables lever 1210to apply force to plate 1230 coupled to force members 1214.

FIGS. 60 and 61 show views of microfluidic cartridge 1216 withself-piercable reservoirs 1228 and mechanical members 1230 for actuatingthe self piercing reservoirs.

FIGS. 62 and 63 show elements of optical detector elements 1220including light sources 1232 (for example, light emitting diodes),lenses 1234, light detectors 1236 (for example, photodiodes) and filters1238. The filters can be, for example, bandpass filters, the filters atthe light sources corresponding to the absorption band of one or morefluorogenic probes and the filters at the detectors corresponding to theemission band of the fluorogenic probes.

Example 7: Preparing Retention Member

Carboxylate surface magnetic beads (Sera-Mag Magnetic Carboxylatemodified, Part #3008050250, Seradyn) at a concentration of about 10¹¹mL⁻¹ were activated for 30 minutes using N-hydroxylsuccinimide (NHS) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in a pH 6.1 500 mM2-(N-Morpholinio)-ethanesulfonic acid (MES) buffer solution. Activatedbeads were incubated with 3,000 Da or 300,000 Da average molecularweight poly-L-lysine (PLL). After 2 washes to remove unbound PLL, beadswere ready for use.

Example 8: Microfluidic Cartridge

Referring to FIGS. 64 and 65, a microfluidic cartridge 300 wasfabricated to demonstrate separation of polynucleotides from inhibitors.Cartridge 300 comprises first and second substrate portions 302′, 304′,which respectively comprise first and second layers 302 a′, 302 b′ and304 a′, 304 b′. First and second layers 302 a′, 302 b′ define a channel306′ comprising an inlet 310′ and an outlet 312′. First and secondlayers 304 a′, 304 b′ define a channel 308′ comprising an inlet 314′ andan outlet 316′. First and second substrate portions 302′, 304′ weremated using adhesive 324′ so that outlet 312′ communicated with inlet314′ with a filter 318′ positioned therebetween. A portion of outlet312′ was filed with the activated beads prepared above to provide aprocessing region 320′ comprising a retention member (the beads). Apipette 322′ (FIG. 66) secured by adhesive 326′ facilitated sampleintroduction.

In use, sample introduced via inlet 310′ passed along channel andthrough processing region 320′. Excess sample material passed alongchannel 308′ and exited device 300′ via outlet 316′. Polynucleotideswere preferentially retained by the beads as compared to inhibitors.Once sample had been introduced, additional liquids, e.g., a wash liquidand/or a liquid for use in releasing the retained polynucleotides wereintroduced via inlet 326′.

Example 9: Retention of DNA

Retention of polynucleotides by the poly-L-lysine modified beads ofdevice 300′ was demonstrated by preparing respective devices comprisingprocessing regions having a volume of about 1 μL including about 1000beads. The beads were modified with poly-L-lysine of between about15,000 and 30,000 Da. Each processing region was filled with a liquidcomprising herring sperm DNA (about 20 uL of sample with a concentrationof about 20 mg/mL) thereby placing the beads and liquid in contact.After the liquid and beads had been in contact for 10 minutes, theliquid was removed from each processing region and subjected toquantitative real-time PCR to determine the amount of herring sperm DNApresent in the liquid.

Two controls were performed. First, an otherwise identical processingregion was packed with un-modified beads, i.e., beads that wereidentical with the poly-L-lysine beads except for the activation andpoly-L-lysine incubation steps. The liquid comprising herring sperm DNAwas contacted with these beads, allowed to stand for 10 minutes,removed, and subjected to quantitative real-time PCR. Second, the liquidcomprising the herring sperm DNA (“the unprocessed liquid”) wassubjected to quantitative real-time PCR.

Referring to FIG. 66, the first and second controls exhibitedessentially identical responses indicating the presence of herring spermDNA in the liquid contacted with the unmodified beads and in theunprocessed liquid. The liquid that had contacted the 3,000poly-L-lysine beads exhibited a lower response indicating that themodified beads had retained substantially all of the herring sperm DNA.The PCR response of the liquid that had contacted the 300,000 Dapoly-L-lysine beads exhibited an amplification response that was atleast about 50% greater than for the 3,000 Da beads indicating that thelower molecular weight surface modification was more efficient atretaining the herring sperm DNA.

Example 10: Releasing DNA from Poly-L-Lysine Modified Beads

Devices having processing regions were packed with 3,000 Dapoly-L-lysine modified beads. Liquid comprising polynucleotides obtainedfrom group B streptococci (GBS) was contacted with the beads andincubated for 10 minutes as above for the herring sperm DNA. This liquidhad been obtained by subjecting about 10,000 GBS bacteria in 10 μl of 20mM Tris pH 8, 1 mM EDTA, 1% Triton X-100 buffer to thermal lysing at 97°C. for 3 min.

After 10 minutes, the liquid in contact with the beads was removed byflowing about 10 μl of wash solution (Tris-EDTA pH 8.0 with 1% Triton X100) through the processing region. Subsequently, about 1 μl of 5 mMNaOH solution was added to the processing region. This process left thepacked processing region filled with the NaOH solution in contact withthe beads. The solution in contact with the beads was heated to 95° C.After 5 minutes of heating at 95° C., the solution in contact with thebeads was removed by eluting the processing region with a volume ofsolution equal to three times the void volume of the processing region.

Referring to FIG. 67, five aliquots of solution were subjected toquantitative real-time PCR amplification. Aliquots E1, E2, and E3 eachcontained about 1 μl of liquid. Aliquot L was corresponds to liquid ofthe original sample that had passed through the processing region.Aliquot W was liquid obtained from wash solution without heating.Aliquot E1 corresponds to the dead volume of device 300, about equal tothe volume of channel 308. Thus, liquid of aliquot E1 was present inchannel 308 and not in contact with the beads during heating. Thisliquid had passed through the processing region prior to heating.Aliquot E2 comprises liquid that was present within the processingregion and in contact with the beads during heating. Aliquot E3comprises liquid used to remove aliquot E2 from the processing region.

As seen in FIG. 67, more than 65% of the GBS DNA present in the initialsample was retained by and released from the beads (Aliquot E2). AliquotE2 also demonstrates the release of more than 80% of the DNA that hadbeen retained by the beads. Less than about 18% of the GBS DNA passedthrough the processing region without being captured. The wash solutionwithout heating comprised less than 5% of the GBS DNA (Aliquot W).

Example 11: Separation of Polynucleotides and Inhibitors

Buccal cells from the lining of the cheeks provide a source of humangenetic material (DNA) that may be used for single nucleotidepolymorphism (SNP) detection. A sample comprising buccal cells wassubjected to thermal lysing to release DNA from within the cells. Device300 was used to separate the DNA from concomitant inhibitors asdescribed above. A cleaned-up sample corresponding to aliquot E2 of FIG.67 was subjected to polymerase chain reaction. A control or crude sampleas obtained from the thermal lysing was also amplified.

Referring to FIG. 68, the cleaned-up sample exhibited substantiallyhigher PCR response in fewer cycles than did the control sample. Forexample, the clean-up sample exceeded a response of 20 within 32 cycleswhereas the control sample required about 45 cycles to achieve thesample response.

Blood acts as a sample matrix in variety of diagnostic tests includingdetection of infectious disease agents, cancer markers and other geneticmarkers. Hemoglobin present in blood samples is a documented potentinhibitor of PCR. Two 5 ml blood samples were lysed in 20 mM Tris pH 8,1 mM EDTA, 1% SDS buffer and introduced to respective devices 300, whichwere operated as described above to prepare two clean-up samples. Athird 5 ml blood sample was lysed and prepared using a commercial DNAextraction method Puregene, Gentra-Systems, MN. The respectivecleaned-up samples and sample subjected to the commercial extractionmethod were used for a Allelic discrimination analysis (CYP2D6*4reagents, Applied Biosystems, CA). Each sample contained an amount ofDNA corresponding to about 1 ml of blood.

Referring to FIG. 69, the cleaned-up and commercially extracted samplesexhibited similar PCR response demonstrating that the processing regionof device 300′ efficiently removed inhibitors from the blood samples.

Example 12: Protease Resistant Retention Member

The preparation of polynucleotide samples for further processing oftenincludes subjecting the samples to protease treatment in which aprotease cleaves peptide bonds of proteins in the sample. An exemplaryprotease is pronase, a mixture of endo- and exo-proteases. Pronasecleaves most peptide bonds. Certain ligands, such as poly-L-lysine canbe susceptible to rupture by pronase and other proteases. Thus, samplesare generally not subjected to protease treatment in the presence of theretention member if the ligands bound thereto are susceptible to theproteases.

Poly-D-lysine, the dextro enantiomer of poly-lysine resists cleavage bypronase and other proteases. The ability of a retention membercomprising bound poly-D-lysine to retain DNA even when subjected to aprotease treatment was studied.

Eight (8) samples were prepared. A first group of 4 samples contained1000 GBS cells in 10 μl buffer. A second group of 4 samples contained100 GBS cells in 10 μl buffer. Each of the 8 samples was heated to 97°C. for 3 min to lyse the GBS cells. Four (4) sample sets were createdfrom the heated samples. Each sample set contained 1 sample from each ofthe first and second groups. The samples of each sample sets weretreated as follows.

Referring to FIG. 70A, the samples of sample set 1 were subjected topronase incubation to prepare respective protein cleaved samples, whichwere then heated to inactivate the proteases. The protein-cleaved,heated samples were contacted with respective retention members eachcomprising a set of poly-L-lysine modified beads. After 5 minutes, therespective sets of beads were washed with 5 microliters of a 5 mM NaOHsolution to separate inhibitors and products of protein cleavage fromthe bound DNA. The respective sets of beads were each contacted with asecond aliquot of NaOH solution and heated to 80° C. for 2 minutes torelease the DNA. The solutions with released DNA were neutralized withan equal volume of buffer. The neutralized solutions were analyzed todetermine the efficiency of DNA recovery. The results were averaged andshown in FIG. 70B.

The samples of sample set 2 were subjected to pronase incubation toprepare respective protein cleaved samples, which were then heated toinactivate the proteases. The protein-cleaved, heated samples werecontacted with respective retention members each comprising a set ofpoly-D-lysine modified beads. After 5 minutes, the respective sets ofbeads were washed with 5 microliters of a 5 mM NaOH solution to separateinhibitors and products of protein cleavage from the bound DNA. Therespective sets of beads were each contacted with a second aliquot ofNaOH solution and heated to 80 (eighty) ° C. for 2 minutes to releasethe DNA. The solutions with released DNA were neutralized with an equalvolume of buffer. The neutralized solutions were analyzed to determinethe efficiency of DNA recovery. The results were averaged and shown inFIG. 70B.

The samples of sample set 3 were subjected to pronase incubation toprepare respective protein cleaved samples. The proteases were notdeactivated either thermally or chemically. The protein-cleaved sampleswere contacted with respective retention members each comprising a setof poly-L-lysine modified beads. After 5 minutes, the respective sets ofbeads were washed with 5 microliters of a 5 mM NaOH solution to separateinhibitors and products of protein cleavage from the bound DNA. Therespective sets of beads were each contacted with a second aliquot ofNaOH solution and heated to 80 (eighty) ° C. for 2 minutes to releasethe DNA. The solutions with released polynucleotides were eachneutralized with an equal volume of buffer. The neutralized solutionswere analyzed to determine the efficiency of DNA recovery. The resultswere averaged and shown in FIG. 70B.

The samples of sample set 4 were subjected to pronase incubation toprepare respective protein cleaved samples. The proteases were notdeactivated either thermally or chemically. The protein-cleaved sampleswere contacted with respective retention members each comprising a setof poly-D-lysine modified beads. After 5 minutes, the respective sets ofbeads were washed with 5 microliters of a 5 mM NaOH solution to separateinhibitors and products of protein cleavage from the bound DNA. Therespective sets of beads were each contacted with a second aliquot ofNaOH solution and heated to 80 (eighty) ° C. for 2 minutes to releasethe DNA. The solutions with released polynucleotides were eachneutralized with an equal volume of buffer. The neutralized solutionswere analyzed to determine the efficiency of DNA recovery. The resultswere averaged and shown in FIG. 70B.

As seen in FIG. 70B, an average of more than 80% of DNA from the GBScells was recovered using sample set 4 in which the samples werecontacted with poly-D-lysine modified beads and subjected to pronaseincubation in the presence of the beads without protease inactivation.The recovery efficiency for sample set 4 can be more than twice as highas for any of the other samples. Specifically, the recovery efficienciesfor sample sets 1, 2, 3, and 4, were 29%, 32%, 14%, and 81.5%,respectively. The efficiencies demonstrate that high recoveryefficiencies can be obtained for samples subjected to proteaseincubation in the presence of a retention member that retains DNA.

Example 13: Operator's Manual for Apparatus for PolynucleotideProcessing

This non-limiting example describes, in the form of an operator'smanual, various embodiments of the claimed apparatus, microfluidiccartridge, kit, methods, and computer program product, in particulardirected to a single cartridge system including a microfluidic PCR assayfor the qualitative detection of microorganisms, such as Group BStreptococcus (GBS). Further descriptions pertinent to GBS analysis arepresented in Example 14.

Sample Preparation Kit, which May Include

-   -   Sample vial containing buffer and preservative    -   Collection Swab with instructions for patient self-collection.    -   A number (e.g., 25) of syringes with filter    -   Vials containing buffer.    -   Patient ID labels for the collection vials    -   3 cc syringes.    -   Microfluidic cartridge, as further described herein.        External Control Sample Kits, which May Include

Positive Control Swab specimen containing Limit of Detection sample of,e.g., GBS bacteria in dehydrated form;

-   -   Vials containing buffer with POSITIVE identification on vial;    -   Vials containing buffer with NEGATIVE identification on vial;        and    -   Syringes.

Equipment:

System with Barcode reader, both of which as further described herein,fitted with, e.g., a 115V or 220V power cord.

Additional Optional Equipment:

-   -   Printer with USB connection    -   Hospital Network connection

Exemplary Use & Indications for Use

The description in this example, is suitable to test samples forpresence of GBS, further details of which are provided in Example 14.

Explanation of Test Application

The apparatus and materials can be used to test for a variety ofpathogens and microorganisms, as further described herein. One exampleis to test for GBS, as further described in Example 14. The tests can beperformed in the near-patient setting by clinicians who are notextensively trained in laboratory procedures. A QC routine can be builtinto the User Interface of the system in order provide continued QualityAssurance for the GBS test. The test can also be performed in a centralhospital “stat” laboratory, provided that the sample testing occurswithin the time-frame required by the physician requesting the test.

Exemplary Warnings & Precautions

Other warnings and precautions specific to GBS are presented in Example14.

-   -   In some embodiments, if a patient is currently being treated        with antibiotics, the test may be a unreliable indicator of a        disease state.    -   Typically, avoid use of the cartridge/sample kit beyond        expiration date.    -   Typically, avoid using cartridge that was previously opened. Air        & moisture exposure can degrade the reagents. Avoid opening the        cartridge until after the sample is ready to be injected.    -   Typically, use new syringe materials for sample preparation.    -   Typically, use swabs and buffer provided in test kit. In some        embodiments, other brands of collection swabs may interfere with        the test performance    -   When a specimen must be stored, refrigeration at 4° C. for a        duration of up to 24 hours is typically indicated.    -   Typically, use protective clothing and disposable gloves while        handling the components of the system and specimen.    -   Typically, use aseptic technique. It can be especially important        to use new disposable gloves to avoid contamination of the test        specimen or test materials.    -   Typically, avoid injecting the sample with high pressure. Gentle        injection is preferred.    -   Typically, handle specimens using Universal Precautions in        accordance with safe hospital procedures for potentially        infectious specimens.    -   Typically, use recommended cleaning agents when cleaning the        system.    -   Typically, avoid cleaning the test cartridge with any cleaning        chemical if spill occurs. If necessary, use a dry lab tissue to        remove liquids.

Typical Storage and Stability Conditions

Storage/Use Transport Product Description Condition Stability conditionSystem 15-35° C., NA −10° C. to 65° C. operational range GBS SamplingKits  4-40° C. Expiration date Temp > 4° C. Do not freeze. Patient or QCSpecimen @15-30° C.  8 hours in buffer (wet) Patient Specimen or QC @4°C. 24 hours in buffer (wet) Patient Specimen in @15-30° C. 24 hourscontainer (dry) Patient Specimen in @4° C. container (dry) GBSCar-fridges- 4-30° C. Do Expiration date Temp > 4° C. unopened notfreeze, GBS Cartridges- Do not use. 60 minutes unopened maximum GBSControl Kits 15-30° C. Expiration date Protection TBD

Exemplary Specimen Collection

-   -   Precaution: Avoid touching Dacron® end of swab with fingers.    -   Remove swab from packaging    -   Wipe away excess vaginal secretions.    -   Insert swab 2 cm into vagina. (front passage).    -   Insert same swab 1 cm into anus. (back passage)    -   Place swab in transport package if stored dry.

Exemplary Specimen Preparation

-   -   Use aseptic technique when handling test materials and specimen.    -   Confirm sample buffer is not expired.    -   Dip swab vigorously 20 times into vial containing 1 cc buffer    -   Remove and discard swab.    -   Label vial with Patient ID and time of collection, if required        by clinical standard procedure.    -   Precaution: Avoid opening cartridge package until ready to use.        Onboard reagents may be sensitive to light and moisture.

Exemplary System Operation Instructions

-   -   System Ready screen may be shown.    -   Touch the screen to stop the screen saver.    -   Raise the Handle and Open Lid of the System to begin.    -   A self-test of the system may begin once the lid is fully opened        and an “open” indicator, if present, is on.

Inspect the system for any used cartridges remaining from a previoustest.

Log-in with User ID and Password.

Scan sample collection vial bar-code.

Open test cartridge. Scan cartridge barcode.

System may present an error message if materials are expired and/or ifspecimen vial and test materials are not appropriately matched Confirmsample ID with patient information.

Enter the patient ID and other hospital identifying information, ifrequested.

Exemplary Instructions for Transferring Sample to Cartridge:

-   -   Put on a new pair of gloves.    -   Place sample vial on counter.    -   Attach tip onto syringe. Draw entire sample into syringe. Draw        in additional 2 cc of air into the syringe.    -   Remove tip and attach filter.    -   Inject. 1 cc sample into cartridge using syringe and an        additional 2 cc air.    -   Precaution: use gentle pressure to inject sample to avoid        backsplash from sample.    -   Exemplary instructions for performing test:    -   Gently rock the cartridge back and forth until pellets are        dissolved.    -   Place Cartridge on the System    -   Close Cover of the System    -   (Test may begin automatically)    -   Results    -   When test is complete, results may be available for display or        print out.    -   Disposal of Materials: Used Cartridge and collection kit can be        treated as biohazard

Preparing Materials for Shipment

Cartridge should typically be packaged in biohazard protective materialsas described by the International Standards.

Exemplary Cleaning Procedure for System

A solution of 10% bleach (0.5% sodium hypochlorite), followed by a cleanwater rinse can be used to disinfect as well as reduced potential DNAcontamination.

DNA contamination can typically be accomplished by cleaning with bleachor materials suitable for eliminating DNA contamination. Chemicon™Nucleic acid removers can also be used to be used after cleaning withordinary disinfectants.

Typically, alcohol or ordinary sanitary wipes will not reduce DNAcontamination of the instrument.

Exemplary Reagent Lot Verification

Purpose—Assessment of cartridge and sample kit lots and verification oftotal system performance.

Recommendation: Upon receipt of a new lot of cartridges or sample kits,a quality control set can be run to confirm, for example, whether thereagent set includes (1) external control and (1) negative externalcontrol.

Exemplary Quality Control Routine

The external positive control serves to monitor and calibrate thesensitivity of the specimen preparation steps and the assay, and can beused to minimize the risk of false negative results. If the test fails,it may invalidate the results from that batch of cartridges, and shouldbe reported to the supplier.

Purpose—Verification of total system performance including assessment ofsample handling technique. Test can be performed at a User selectedpreset interval.

Recommendation: When test is performed, run a QC set including (1)Positive external control and (1) Negative external control, If QC testfails to yield expected results, contact manufacturer.

Exemplary Quality Control Routine Directions

-   -   Go to Quality Control Screen    -   Enter User ID    -   Select QC Tech verification, New Reagent Lot, Daily QC Run        samples as directed on screens.

System Self-Test

The System may perform a System initiation test when it is powered up.As the Start Up Routine of each Patient Test Sample or QC Sample, theSystem may run a self-test to determine if, for example, theelectronics, optical system, heaters and temperature sensors arefunctioning as intended.

Exemplary Internal Controls (On-Cartridge)

Reagents which can be used in the assay may be included on the cartridgeto reduce the potential for user handling errors and contamination. Twotypes of on-cartridge positive and negative controls strategies can beincorporated within each microfluidic cartridge to monitor individualPCR assay performance. Two examples are:

1) Positive Internal Control Plasmid (ICP):

An internal control plasmid can provide a control for integrity of thePCR assay reagents as well as being an indicator for presence of PCRinhibitory substances in the specimen, i.e. it can be a control forfalse negative results. During thermocycling, amplification of thisregion may produce a distinct fluorogenic signal in both PCR lanes.Failure to amplify the internal control sequence, in the absence of apositive sample, can be indicative of either a failure of the reagentmix or presence of PCR inhibitors in the specimen. This can invalidatethe results of the test as indicated by an error code on the instrument.“IND”. can be displayed to report an indeterminate result.

2) Negative Internal Control PCR Lane:

A parallel lane can be used to run a second PCR with reagents from thesame mix without any sample. This can provide a control against falsepositive results due to contamination of the reagents. A failed resultin the negative control lane can invalidate the result and can beindicated by an error code on the instrument. “IND” can be displayed toreport an indeterminate result.

Exemplary Real Time PCR for GBS DNA Detection

In various embodiments, the test can use real-time Polymerase ChainReaction (PCR) for the amplification of a cfb gene sequence of GBSrecovered from clinical samples and fluorogenic target-specifichybridization for the detection of the amplified DNA. The cfb geneencodes the CAMP factor, a diffusible extracellular protein which istypically present in GBS isolates. The Group B Streptococcus (GBS)detection test can be an integrated, raw-sample-to-result type ofnucleic acid amplification assay. A TaqMan fluorogenic probe may be usedto detect the PCR amplicons. Reagents used in the assay may be includedon the cartridge to reduce potential for user handling errors andcontamination.

Exemplary Potential Precautions

In some embodiments, the test system may not be qualified foridentifying targets such as GBS DNA, in specimens other than vaginaland/or rectal specimens. Urine and Blood specimens may not be qualified.

In some embodiments, a patient undergoing antibiotic treatment may notbe able to obtain a correct diagnosis with this or other diagnostictests.

In some embodiments, the test may not yield a GBS culture suitable fordirect identification of the bacteria by a microbiologist. In someembodiments, the test may not provide susceptibility results that areneeded to recommend a treatment for penicillin-allergic persons.

Exemplary Potential Interfering Substances

In some embodiments, urine and vaginal secretions if present in verylarge quantities, may interfere with the test.

Typically, contaminants such as blood, meconium, and amniotic fluidcontamination of the samples is not likely to interfere with the test.

Typically, drug interference (such as present in vaginal and rectalsecretions), other than antibiotics, are not known at this time tointerfere with PCR.

Exemplary Performance Characteristics and Interpretation

The flow chart in FIG. 71 outlines an exemplary set of criteria whichmay be used by the decision algorithm in the instrument to interpret theresults. PCR reactions (Sample and Control) may be interpreted aspositive or negative for the target in question. A logical algorithm maybe used to determine if sample is definitively Positive or Negative orIndeterminate.

Exemplary Cross Reacting Substances

Specificity of the primers and probes can be tested with real-time PCR(Taqman assay) using genomic DNAs isolated from the following organisms:nine GBS serotypes (serotype 1a, 1b, 1c, II, III, IV, V, VI and VII;American Type Culture Collection and National Center for Streptococcus,Canada); 10 clinical GBS isolates; 60 clinical samples; a wide varietyof gram-positive and gram-negative bacterial strains as well as twoyeast strains and RSV type I and 2.

Exemplary Microorganisms

PATHOGEN TYPE Pseudomones aeruginosa Gram − Bacteria Proteus mirabilisGram − Bacteria Kiebsiella oxytoca Gram − Bacteria Kiebsieflapneurnoniae Gram − Bacteria Escherichia cot (clinical isolate 1) Gram −Bacteria Escherichia coli (clinical isolate 2) Gram − BacteriaAcinetobacter baumannd Gram − Bacteria Serra. marcescens Gram − BacteriaEntembacter aerugenes Gram + Bacteria Enterococcus Maclean Gram +Bacteria Staphylococcus aureus (clinical isolate 1) Gram + BacteriaStaphylococcus aureus (clinical isolate 2) Gram + Bacteria Streptococcuspyogenes Gram + Bacteria Streptococcus viridans Gram + Bacteria Listenamonocytogenes Gram + Bacteria Enterococcus sps. Gram + Bacteria Cendidaglabrata Yeast Candida albicans Yeast Streptococcus Group C Gram +Bacteria Streptococcus Group G Gram + Bacteria Streptococcus Group FGram + Bacteria Enterococcus feecalis Gram + Bacteria Streptococcuspneumoniae Gram + Bacteria Staphylococcus epiderrnidis (C-) Gram +Bacteria Gardenerella vaginalis Gram + Bacteria Micrococcus spa Gram +Bacteria Haemophilus influenza° Gram − Bacteria Neisseria gonorrhoeaeGram − Bacteria Moraxella catarrahlis Gram − Bacteria Salmonella sps.Gram − Bacteria Chlamytha trechomatis Gram − Bacteria Peptostreptococcusproduct. Gram + Bacteria Peptostreptococcus anaerobe. Gram + BacteriaLactobacillus lennentum Gram + Bacteria Eubacterium lentum Gram +Bacteria Herpes Simplex Virus I (HSV I) Virus Herpes Simplex Virus ll(HSV II) Virus

Exemplary Troubleshooting Chart

Problem Possible Cause Possible Action Positive Control reads Sample notReview sampling method. negative re-suit. No other processed Retest.indication. adequately. Bulk Lysis not Test new lot of cartridge.performed, re- Review storage location of agents degraded. cartridge(<30 C.). Contact HandyLab. Negative Control reads Contamination CleanInstrument. Review positive. No other sampling method. Retest.indication. Error: IND (indeterminant IC fail, PCR not Test new lot ofcartridge. result-IC fail) performed. Error: IND (indeterminantBorderline result- Retest with new patient result) IC passed. sample.

Example 14: Operator's Manual for Apparatus for PolynucleotideProcessing

This non-limiting example describes, in the form of user instructions,various embodiments of the claimed apparatus, microfluidic cartridge,kit, methods, and computer program product, in particular directed to amicrofluidic cartridge for use in a microfluidic PCR assays includingthe qualitative detection of microorganisms such as Group BStreptococcus.

Presence of Group B Streptococci (GBS) remains a leading cause ofserious neonatal infection despite great progress in prevention sincethe 1990's with sepsis, pneumonia, and meningitis affecting the babyafter birth. The GBS test system can be used for the rapid, qualitativedetection of Group B streptococcus (GBS) DNA in vaginal/rectal samples.

Exemplary Use and Indications for Use

In various embodiments, the GBS test system can be used for the rapid,qualitative detection of microorganisms in clinical samples, such asGroup B streptococcus (GBS) DNA in vaginal/rectal samples.

Typical indications for use of the GBS test include, for example, arapid screening test in the prenatal care regimen for the maternitypatient to determine the need for antibiotic treatment during labor, asdescribed by the CDC guidelines (Centers for Disease Control andPrevention. Prevention of Perinatal Group B Streptococcal Disease:Revised Guideline from CDC. Morbidity and Mortality Weekly Report, Aug.16, 2002; 51 (No. RR-11); 1-24). The test can provide rapid results atthe point of care or in a central laboratory with rapid turnaroundservice during the intrapartum and prepartum phase of the maternitypatient. The test can also be used to detect GBS DNA in vaginal orrectal samples of any subject suspected of GBS infection. See also MarkA. Burns, Brian N. Johnson, Sundaresh N. Brahmasandra, Kalyan Handique,James R. Webster, Madhavi Krishnan, Timothy S. Sammarco, Piu M. Man,Darren Jones, Dylan Heldsinger, Carlos H. Mastrangelo, David T. Burke“An Integrated Nanoliter DNA Analysis Device” Science, Vol. 282, 16 Oct.1998.

Explanation of Test Application

The Center for Disease Control and Prevention recommends universalprenatal screening for vaginal/rectal GBS colonization of all women at35-37 weeks gestation in order to determine the need for prophylacticantibiotics during labor and delivery. The current CDC recommendation isthe culture-based test method (Standard Culture Method), from whichresults are typically available in 48-72 hours, compared to about 30minutes for the present test. The test can utilize automated samplepreparation and real-time PCR to identify the cfb gene in the GBS genomewhich is an established identification sequence that encodes the CAMPfactor. The CAMP factor is an extra cellular protein typically presentin GBS isolates. The CAMP factor can be used for the presumptiveidentification of GBS bacteria in clinical samples by the culturemethod. This test can be performed in the near-patient setting byclinicians who are not extensively trained in laboratory procedures. AQC routine can be built into the User Interface in order providecontinued Quality Assurance for the GBS test. The test can also beperformed in a central hospital “stat” laboratory provided that thesample testing occurs within the time-frame required by the maternitydepartment.

Possible Contraindications

The GBS test may be contraindicated for persons with allergy topolyester contained in the specimen collection swab.

Exemplary Warnings & Precautions

Warnings herein may be additional to those of general application shownin Example 13, herein.

-   -   GBS test may not provide susceptibility results that are        recommended for penicillin-allergic women.    -   Because IV antibiotics typically need to be started at least 4        hours before delivery, and in the absence of prepartum GBS data,        it may be important to collect samples and start the GBS test as        soon as possible after the patient enters the Labor and Delivery        area of the hospital.    -   In some embodiments, if a patient is currently being treated        with antibiotics, the test may be a unreliable indicator of        disease state.    -   Buffer may contain Sodium Azide as a preservative. Health Hazard        if ingested.    -   Typically, test the specimen within, for example, 8 hours of the        sampling and storage in the buffer. In the situation where a        specimen must be stored, refrigeration at 4 C for a duration of        up to 24 hours may be used.    -   Typically, avoid use of the test materials beyond the expiration        date.    -   Typically, avoid opening the cartridge until after the sample is        ready to be injected.    -   Typically, avoid using a cartridge if the protective, metallized        bag is opened. In some embodiments, light, air and moisture        exposure may degrade the reagents.    -   Typically, use swabs, syringe and buffer provided in test kit.        In some embodiments, other brands of GBS collection swab may        interfere with the test performance.    -   Typically, materials are single use only; reusing materials may        give erroneous results.    -   Avoid opening cartridge package until ready to test. Onboard        reagents may be sensitive to light and moisture. Avoid use if        package seal is broken and foil pouch is no longer expanded        (puffy).

Exemplary Sample Collection Kit

-   A. Swabs-   B. Tubes containing sample collection buffer.-   C. Cannula tips-   D. Syringes (e.g., 3 cc)-   E. Syringe filters-   F. GBS microfluidic cartridge

Exemplary Specimen Collection & Buffer Suspension Instructions

-   -   Caution: Typically, use only collection kit; avoid touching end        of swab with fingers.    -   Remove swab from packaging.    -   Wipe away excess vaginal secretions.    -   Insert swab 2 cm into vagina (front passage).    -   Insert same swab 1 cm into anus (back passage).    -   Confirm Vial of Sample buffer (B) is not expired.    -   Dip swab (A) vigorously up and down 20 times into vial        containing buffer.    -   Remove and discard swab.    -   Label vial with patient ID information

Exemplary Preparation of Sample for Testing

-   -   Cannula tip (C) can be attached to the syringe (D)    -   Draw some or all of sample into syringe.    -   May draw in additional air (e.g., 2 ml).    -   Cannula tip (C) may be replaced with filter (E).    -   Cartridge Package can be opened at the tear points marked on the        package.    -   Barcodes of the Sample vial (B) and Cartridge (F) can be scanned        with the System scanner. System may warn if materials are        expired.    -   Patient identification information can be entered, if needed.    -   Can lay cartridge on flat surface or hold it flat with luer in        upright position. Typically, assure that the the cartridge        remains label side up during the procedure.    -   Sample (including excess air) can be injected into cartridge        using syringe/filter assembly. Gentle injection pressure can be        used to avoid backsplash from sample.    -   Syringe/filter assembly can be removed from cartridge.    -   Cartridge can be gently rocked side-to-side about 10 times until        the pellets inside the sample chamber are dissolved and mixed.    -   Cartridge can be placed on the System    -   Cover of the System can be closed and handle can be locked in        down position. (Test may begin automatically)

Results

When test is complete, results may be clearly displayed. Results can beprinted or stored as determined by lab procedures.

Exemplary Disposal of Materials

Cartridge and collection kit should be treated as biohazard.

Exemplary Recommended Lab Quality Control Routine Exemplary TestVerification

On a weekly basis, (1) positive external control and (1) negativeexternal control can be run. A QC set to confirm system total systemperformance. This procedure is also recommended when training new users.

Exemplary Quality Control Routine Directions:

May run samples as directed on display screens. If a QC test fails toyield expected results, manufacturer can be contacted.

The external positive control can include a lyophilized aliquot ofStreptococcus agalactiae (GBS) cells that are reconstituted at run-timewith sample collection buffer. The number of GBS cells in the externalpositive control may be approximately equal to the Minimum DetectableLimit (MDL) of the test.

Exemplary quality control tests also include an System Self-test QC, asdescribed in Example 14.

Exemplary Internal Controls (On-Cartridge)

Typical reagents for the assay may be included on the cartridge toreduce the potential for user handling errors and contamination. Twotypes of on-cartridge positive and negative controls strategies may beincorporated within each microfluidic cartridge to monitor individualPCR assay performance.

An exemplary positive internal control plasmid for GBS is a doublestranded circular DNA molecule containing a 96 bp region comprised of aunique 39 bp artificial DNA sequence flanked by the forward and reversesequences from the cfb gene can be included in the lyophilized mastermix along with a second distinct fluorogenic probe specific to thisunique sequence. The Failure to amplify the internal control sequence,in the absence of a positive GBS sample, can be indicative of either afailure of the reagent mix or presence of PCR inhibitors in thespecimen.

Exemplary Real Time PCR for GBS DNA Detection

The test can utilize real-time Polymerase Chain Reaction (PCR) for theamplification of a cfb gene sequence of GBS recovered from clinicalsamples. A fluorogenic target-specific Tacman® probe can be used for thedetection of the amplified DNA. The cfb gene encodes the CAMP factor, adiffusible extracellular protein which is typically present in GBSisolates. The Group B Streptococcus (GBS) detection test may be anintegrated, raw-sample-to-result type of nucleic acid amplificationassay. Typical reagents for the assay can be included on the cartridgeto reduce potential for user handling errors and cross contamination.

Exemplary Possible Test Limitations

In some embodiments, the test system may not be qualified foridentifying GBS DNA in specimens other than vaginal/rectal specimens.For example, urine and blood specimens may not be qualified in someembodiments.

In some embodiments, a patient undergoing antibiotic treatment may notbe able to obtain a correct GBS diagnosis.

In some embodiments, the test may not yield a GBS culture suitable fordirect identification of the bacteria by a microbiologist.

Exemplary Performance Characteristics and Interpretation

10-30% of pregnant women are colonized with GBS. The GBS colonizationcut-off is determined to be approximately 1000 copies of DNA/sampledetermined by amplification of the cfb gene sequence. The user isreferred to the flow-chart of FIG. 71 for application of exemplarycriteria for result interpretation, where the target in question is GBSand IC plasmid.

Exemplary Possible Interfering Substances

In some embodiments, urine or vaginal secretions, or mucus, if presentin large quantities, may interfere with test.

In some embodiments, blood, meconium, or amniotic fluid contamination ofthe samples is typically not likely to interfere with the test.

In some embodiments, drug interference (present in vaginal and rectalsecretions), other than antibiotics, are typically not known at thistime to interfere with PCR.

Exemplary Results Interpretation and Expected Values

10-30% of pregnant women can be colonized with GBS. The GBS colonizationcut-off can be determined to be approximately. 1000 copies of DNA/sampledetermined by amplification of the cfb gene sequence.

PCR reactions can be interpreted as positive or negative for GBS andInternal control. A logical algorithm may be used to determine if sampleis Positive or Negative or Indeterminate.

Exemplary Storage and Stability Information

Description Storage/Use Condition Stability Patient Specimen in buffer(wet) @15-30° C.  8 hours Patient Specimen in buffer (wet) @4° C. 24hours Patient Specimen (dry storage) @15-30° C. 24 hours GBSCartridges-unopened 4-30° C. Expiration date GBS Cartridges-opened Donot use. 60 minutes max.

Each reference cited herein is incorporated by reference in itsentirety, including: U.S. Pat. Nos. 6,057,149, 6,048,734, 6,130,098,6,271,021, 6,911,183, CA2,294,819, U.S. Pat. Nos. 6,575,188, 6,692,700,6,852,287, and Canadian patent application no. CA 2,294,819.

A number of embodiments of the technology have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the technology.Accordingly, other embodiments are within the scope of the followingclaims.

1. (canceled)
 2. An apparatus for processing and amplifying a pluralityof polynucleotide-containing samples a multi-lane microfluidiccartridge, the apparatus comprising: a reagent reservoir; a receivingbay configured to receive a multi-lane microfluidic cartridge configuredto process the plurality of polynucleotide-containing samplesindependently of each other, the multi-lane microfluidic cartridgecomprising a plurality of sample lanes configured to share a reagentreceived in the multi-lane microfluidic cartridge from the reagentreservoir, each sample lane of the plurality of sample lanes comprisinga dedicated pipette inlet and an amplification reaction zone, thededicated pipette inlets of adjacent lanes spaced apart from oneanother, the multi-lane microfluidic cartridge further configured topermit samples to be loaded into the multi-lane microfluidic cartridgeat different times, and passed to the amplification reaction zoneindependently of one another, a sample lane of the multi-lanemicrofluidic cartridge further comprising a processing chamber; thereceiving bay comprising a first heat source configured to thermallycouple to the multi-lane microfluidic cartridge when the multi-lanemicrofluidic cartridge is received in the receiving bay, the first heatsource configured to apply heat to one or more selected regions of thecartridge at one or more selected times, in order to prepare one or moreof the polynucleotides for amplification, the first heat sourceconfigured to apply heat to the processing chamber when the multi-lanemicrofluidic cartridge is received in the receiving bay; a magnetconfigured to move into and out of place to apply a magnetic field to apolynucleotide-loaded retention member in the processing chamber of themulti-lane microfluidic cartridge when the multi-lane microfluidiccartridge is received in the receiving bay; the receiving bay furthercomprising a plurality of second heat sources configured to align withand thermally couple to one or more selected regions of the multi-lanemicrofluidic cartridge when the multi-lane microfluidic cartridge isreceived in the receiving bay, the plurality of second heat sourcesconfigured to apply heat to the one or more selected regions of thecartridge at one or more selected times, in order to amplify one or moreof the polynucleotides, wherein the plurality of second heat sources areconfigured to cyclically heat the second heat sources in a series ofheating phases, wherein each heating phase comprises each of theplurality of second heat sources being cycled between at least twotemperatures, wherein each of the plurality of second heat sources isconfigured to maintain a substantially uniform temperature in anamplification reaction zone thermally coupled to the second heat sourceat each temperature of the at least two temperatures when the multi-lanemicrofluidic cartridge is received in the receiving bay, and wherein theplurality of second heat sources are configured to perform independentamplification reactions in the plurality of sample lanes when themulti-lane microfluidic cartridge is received in the receiving bay; andan optical detector configured to detect the presence of the one or moreamplified polynucleotides in the amplification reaction zone of a laneof the multi-lane microfluidic cartridge, the optical detectorcomprising a read head that does not cover the dedicated pipette inletsof the multi-lane microfluidic cartridge when the multi-lanemicrofluidic cartridge is received in the receiving bay, therebypermitting loading of separate samples while other samples areundergoing cyclical heating.
 3. The apparatus of claim 2, wherein thereagent reservoir comprises a wash buffer reservoir, and wherein themulti-lane microfluidic cartridge comprises a plurality of sample lanesconfigured to share a wash buffer received in the multi-lanemicrofluidic from the wash buffer reservoir.
 4. The apparatus of claim2, wherein the reagent reservoir comprises a release reagent reservoir,and wherein the multi-lane microfluidic cartridge comprises a pluralityof sample lanes configured to share a release reagent received in themulti-lane microfluidic cartridge from the release reagent reservoir. 5.The apparatus of claim 4, wherein the release reagent reservoircomprises a reservoir comprising a hydroxide solution, and wherein themulti-lane microfluidic cartridge comprises a plurality of sample lanesconfigured to share the hydroxide solution received in the multi-lanemicrofluidic from the release reagent reservoir.
 6. The apparatus ofclaim 2, further comprising the multi-lane microfluidic cartridgereceived in the receiving bay and thermally coupled to the first heatsource and the plurality of second heat sources.
 7. The apparatus ofclaim 6, wherein each sample lane of the plurality of sample lanescomprises a sample inlet valve in fluidic communication with thededicated pipette inlet.
 8. The apparatus of claim 2, wherein the firstheat source is configured to release one or more polynucleotides from apolynucleotide-loaded retention member in contact with a release reagentin a sample lane of the plurality of sample lanes when the multi-lanemicrofluidic cartridge is received in the receiving bay.
 9. Theapparatus of claim 2, wherein each of the plurality of second heatsources comprises a plurality of resistive heaters.
 10. The apparatus ofclaim 9, wherein the plurality of resistive heaters is configured tomaintain a substantially uniform temperature in an amplificationreaction zone thermally coupled to the plurality of resistive heaters ateach temperature of the at least two temperatures when the multi-lanemicrofluidic cartridge is received in the receiving bay.
 11. Theapparatus of claim 10, wherein each of the plurality of resistiveheaters is fixed in position relative to the multi-lane microfluidiccartridge during cyclical heating.
 12. The apparatus of claim 2, whereinthe optical detector comprises a light source configured to emit lightin an absorption band of a fluorescent dye into the amplificationreaction zone of each lane of the multi-lane microfluidic cartridge whenthe multi-lane microfluidic cartridge is received in the receiving bay,and a light detector configured to detect light in an emission band ofthe fluorescent dye from the amplification reaction zone of each lane ofthe multi-lane microfluidic cartridge when the multi-lane microfluidiccartridge is received in the receiving bay, and wherein the fluorescentdye binds to a fluorescent polynucleotide probe or a fragment thereof.13. The apparatus of claim 12, wherein the optical detector isconfigured to selectively emit light in the absorption band of thefluorescent dye, and selectively detect light in the emission band ofthe fluorescent dye.
 14. The apparatus of claim 6, wherein themulti-lane microfluidic cartridge comprises one or more componentsconfigured to inhibit motion of a material in the multi-lanemicrofluidic cartridge when the multi-lane microfluidic cartridge isreceived in the receiving bay.
 15. The apparatus of claim 14, whereinthe one or more components configured to inhibit motion of a materialcomprises a valve configured to transform from an open to a closed statewhen the multi-lane microfluidic cartridge is received in the receivingbay.
 16. The apparatus of claim 15, where the valve is configured toallow the material to pass along a channel from a position on one sideof the valve to a position on the other side of the valve, and wherein,upon actuation when the multi-lane microfluidic cartridge is received inthe receiving bay, the valve is configured to transition to a closedstate that prevents the material from passing along the channel from oneside of the valve to the other.
 17. The apparatus of claim 16, whereinthe material comprises a polynucleotide-containing sample.
 18. Theapparatus of claim 17, wherein the one or more components configured toinhibit motion of the polynucleotide-containing sample comprises aplurality of valves configured to isolate an amplification reaction zoneduring cyclical heating when the multi-lane microfluidic cartridge isreceived in the receiving bay.
 19. The apparatus of claim 16, whereinthe material comprises a gas.
 20. The apparatus of claim 16, wherein thematerial comprises a reagent.
 21. The apparatus of claim 6, wherein themulti-lane microfluidic cartridge is disposable.
 22. The apparatus ofclaim 2, wherein the multi-lane microfluidic cartridge comprises a wastereservoir and a channel extending between the processing chamber and thewaste reservoir.
 23. The apparatus of claim 22, wherein the multi-lanemicrofluidic cartridge comprises a valve in the channel extendingbetween the processing chamber and the waste reservoir, the valveconfigured to inhibit motion of a material in the channel when themulti-lane microfluidic cartridge is received in the receiving bay. 24.The apparatus of claim 2, further comprising: a second receiving bayconfigured to receive a second multi-lane microfluidic cartridge; and aprocessor configured to: concurrently process a plurality of samples inthe first multi-lane microfluidic cartridge and the second multi-lanemicrofluidic cartridge when the first and second multi-lane cartridgesare received in the receiving bay and the second receiving bay,respectively, and consecutively process a plurality of samples in thefirst multi-lane microfluidic cartridge and the second multi-lanemicrofluidic cartridge when the first and second multi-lane cartridgesare received in the receiving bay and the second receiving bay,respectively.